Glossary

Actinomycin D results in the cessation of new RNA transcription. Consequently, serial determinations of specific RNA levels will allow one to calculate the mRNA half-life. Variation in mRNA half-life between control and stimulated conditions suggests that expression of a gene of interest is regulated at the level of mRNA stability.

This has been identified as the von Willebrand factor cleaving protease. It is an enzyme of the family identified as “a disintegrin and metalloprotease with thrombospondin type-1 motifs.”

A humanized anti-CD52 monoclonal antibody that depletes lymphoid cells.

A large proportion of Ki-1 positive lymphomas are characterized by a t(2;5). The breakpoint involves nucleophosmin, a ubiquitously expressed gene, and ALK. ALK is a member of the insulin receptor family of transmembrane receptor kinases. which is not normally expressed in hematopoietic tissues. The chimeric mRNA results in a fusion protein that is no longer membrane bound. This fusion protein is thought to contribute to the pathogenesis of the resultant lymphoma.

If the nucleotide basis for a specific genetic abnormality is known, oligonucleotides specific for the wild type and mutant sequences can be designed and used to probe Southern blots of an individual’s genomic DNA. The pattern of hybridization gives specific information regarding which alleles are present. In a polymorphic disease such as b thalassemia (in which multiple mutations can give rise to the same disease phenotype), multiple probes might be required to detect all possible causes. In addition, new mutations causing the same disease would be missed. However, should a specific probe prove useful for one population group or be positive in one family member, that probe becomes very useful for the individual under study.

By using generic PCR primers flanking the immunoglobulin or T cell receptor genes, the precise rearranged gene characteristic of a B or T cell neoplasm can be amplified. The rearranged gene contains somatic mutations that occur as part of the receptor rearrangement process. If the PCR product is then sequenced, new PCR primers can then be designed that are unique to the patient’s tumor. Such allele-specific PCR can then be used to detect blood cell contamination by tumor and to detect minimal residual disease following therapy.

Located on chromosome 21, AML1 (CBFα2, RUNX1) is the fusion partner of ETO in t(8:21). The gene is homologous to the runt gene of Drosophila and encodes a transcription factor. Normal hematopoietic targets of AML1 include CD13, GM-CSF, MPO, IL-3, and the T cell antigen receptor. AML1 binds as a heterodimer, partnered with CBFβ.

Retroviruses whose coat proteins bind to a receptor found throughout multiple species, usually including man, making these vectors suitable for human use. Use in hematologic gene therapy is encumbered by the level of receptor expression on human stem cells.

Specific gene expression can be interrupted by the introduction of short single-stranded deoxyribonucleic acids (ODN) into a cell. Several mechanisms have been postulated to account for these results including interruption of ribosome binding to mRNA, enhanced degradation of mRNA mediated by the double-strand specific RNAseH, DNA triplex formation, and impairment of translation efficiency. Most successful attempts using antisense ODN have targeted sequences surrounding and including the initiation codon. To reduce nuclease attack, the antisense ODN are often synthesized using an altered chemistry involving thiol rather than phosphodiester linkages.

Plasmids encoding the β-galactosidase enzyme are co-transfected with genes of interest in order to track transfection efficiency and to identify individual transfected cells. The presence of β-galactosidase activity in the cytoplasm of transfected cells can be readily detected by its ability to convert a colorless substrate to a blue-colored product. This is usually assayed using a fluorimeter.

These transcriptional proteins are characterized by the ability to dimerize through a domain containing two alpha helical regions separated by a loop structure. Examples of this family of transcription factors include E12/E47 of the immunoglobulin promoter or Myo D of muscle cell regulation.

This gene, located on chromosome 11q13, was first identified as the site of translocation p(11;14)(q13;q32). It has a strong association with central acinar/mantle cell lymphoma and functions in normal cells as the G₁ cyclin, cyclin D₁. Normally, lymphocytes lack cyclin D₁ expression; its aberrant expression resulting from chromosomal translocation of the Bcl-1 locus to an immunoglobulin locus is thought to be associated with aberrant proliferation.

This gene product normally functions to suppress programmed cell death (apoptosis). Its overexpression is associated with the most common molecular abnormality in non-Hodgkin’s lymphoma, t(14;18)(q32;q21), present in 80% of follicular small cleaved cell lymphoma. Presumably, suppression of apoptosis leads to extended cell survival, a characteristic of low-grade lymphomas.

This gene is a member of the IκB family. Presently, it is unclear how this protein acts in tumorigenesis, but it is likely that its involvement in transcriptional processes is critical.

A zinc finger transcription factor, expression of which is altered in approximately one-third of diffuse B large cell lymphomas as a consequence of 3q27 translocations. Its target genes are unknown.

A method that exploits the formation of branched DNA to provide a sensitive and specific assay for viral RNA or DNA. The assay is performed in a microtiter format, in which partially homologous oligodeoxynucleotides bind to target to create a branched DNA. Enzyme-labeled probes are then bound to the branched DNA, and light output from a chemiluminescence substrate is directly proportional to the amount of starting target RNA. Standards provide quantitation. The assay displays a 4 log dynamic range of detection, with greater sensitivity to changes in viral load than RT-PCR-based assays. It has been employed to quantitate levels of HIV, HCV, and HBV.

This gene is located on human chromosome 9 and encodes a tyrosine kinase whose role in normal hematopoiesis is unclear. Fusion of c-abl to the bcr gene on human chromosome 22 occurs during the formation of the Ph1 chromosome in chronic myelogenous leukemia. This fusion eliminates the first two or three exons of c-abl and results in unregulated tyrosine kinase activity. The resultant fusion protein is either 210 kDa or 195 kDa. The latter version is more acutely transforming in experimental settings; it is also associated with acute lymphoblastic leukemia and with a worse prognosis in both disease settings.

A method of transfection hat relies on the production of a calcium/phosphate/DNA microprecipitate, which is then taken up by cells by pinocytosis. The method is very effective for a number of commonly used mammalian cell expression systems including COS, BHK, 293, and CHO cells.

Located on chromosome 9, CAN is part of t(6:9). CAN forms part of the nuclear pore. Because it has two different fusion partners but a consistent phenotype, CAN is likely the critical component of t(6:9).

The bacterial gene for chloramphenicol resistance, CAT is commonly used as a reporter gene for investigating physiologic gene regulation. The assay depends on the ability of transfected genes to drive expression of a CAT-containing vector, resulting in synthesis of the enzyme. Cytoplasmic activity of CAT converts ¹⁴C chloramphenicol to its acetylated form in the presence of acetyl CoA. The acetylated forms are separated from the ¹⁴C substrate using thin layer chromatography.

A family of heterodimeric transcription factors containing a common beta subunit, CBFβ, and one of three CBFα subunits; CBFα1, AML1 (CBFα 2), or CBFα 3. CBFβ increases the affinities of the CBFα subunits for DNA. Mice lacking CBFβ or AML1 fail to develop definitive hematopoiesis.

CBFβ-SMMHC is expressed in the blasts of patients with the FAB M4eo subtype of AML as a consequence of the inv(16)(p13q22) or the t(16;16)(p13q22) chromosomal abnormalities. In CBFβ-SMMHC the CBFβ transcription factor is fused to the tail domain of a smooth muscle myosin heavy chain.

A member of the cell cycle cyclin-dependent protein kinase family.

A pan-T cell marker that also reacts with a range of neoplastic B cells, e.g., B cell chronic lymphocytic leukemia (B-CLL), B cell small lymphocytic lymphoma (B-SLL), and mantle cell lymphoma. CD5 is expressed on all T lymphocyte subsets and is modulated during cellular activation. Aberrant loss of CD5 expression is a characteristic of some T cell lymphomas.

A cell surface enzyme with neutral metalloendopeptidase activity that inactivates a variety of biologically active peptides. CD10 is expressed on the cells of lymphoblastic, Burkitt’s, and follicular germinal center lymphomas, and on cells from patients with chronic myelocytic leukemia (CML). It is also expressed on the surface of normal early lymphoid progenitor cells, immature B cells within adult bone marrow and germinal center B cells within lymphoid tissue.

A co-stimulatory membrane protein belonging to the TNF superfamily. It is expressed on antigen presenting cells and interacts with its counterpart, CD40, which is present on B cells. CD40L-CD40 interaction triggers B cell activation and proliferation, and is therefore essential for the induction of humoral and cellular immunity. The CD40L-CD40 system is also involved in thymic selection. Gene transfer of CD40L into tumor cells elicits an antitumor immune response and suppresses tumor growth, probably by triggering local antigen presenting cells to present tumor antigen to the cellular immune system.

A major sialoglycoprotein expressed on the surface of leukocytes and platelets. CD43 plays an important role both in adhesion and signal transduction. It is involved in T cell/B cell interaction during immune reaction and binds on antigen-presenting B cells.

CD46 is a 45–70 kDa protein that is expressed on every cell and tissue, with the exception of erythrocytes. CD46 downregulates the activation of complement on host tissue. It performs this function by acting as a cofactor that binds to C3b and C4b. It also serves as a receptor for certain viruses.

A 21–28 kD glycopeptide expressed on the surface of nearly all human lymphocytes, monocytes and macrophages. The function of CD52 is uncertain. It is targeted by alemtuzumab (Campath-1H).

The CD80 molecule is expressed on activated B lymphocytes and macrophages, while CD86 is expressed on B lymphocytes, monocytes and dendritic cells. CD80/86 provides an important co-stimulatory signal for T cell activation by interacting with its cognate receptors on T cells, CD28 and cytotoxic T lymphocyte antigen-4 (CTLA-4). Many tumor cells lack B7 expression and as a result do not provide the co-stimulatory signal necessary to trigger T cell activation upon the recognition of tumor antigens. This leads to T cell anergy and is postulated to be one of the main mechanisms responsible for the poor immunogenicity of tumor cells.

A related group of cellular kinases, present in virtually all cells, that are regulated both positively and negatively by specific phosphorylation events and by association with other proteins. Activation of the kinases is dependent on cyclins, present only during certain phases of the cell cycle.

Proteins that inhibit the cyclin-dependent kinases by stoicheometric combination, arresting cells in G₁ phase. The Cdk inhibitors include p27, p21 and the p16 Ink 4A family of proteins. The latter are implicated as tumor suppressor genes, as their deficiency in mice leads to rapid cellular proliferation and a high rate of spontaneous tumor development. Moreover, deficiency of p16 family members has been associated with numerous types of human tumors, including a fraction of cases of B cell ALL and T cell leukemia.

A complementary copy of a stretch of DNA produced by recombinant DNA technology. Usually, cDNA represents the mRNA of a given gene of interest.

An ~62 kDa leucine zipper protein that does not form homodimers, but functions in a heterodimeric complex with c-jun and other members of the AP1 family of transcription factors.

A method for detecting genomic alterations in tumor cells. DNA is extracted from tumor and from normal tissues and differentially labeled with fluorescent dyes. Once the DNA samples are mixed and hybridized to normal metaphase chromosome spreads, chromosomal regions that are under-represented or overrepresented in the tumor sample can be identified. This technique has been modified to allow detection of changes in single genes by array CGH. In array CGH, gene amplifications and/or deletions are detected by competitive differential hybridization of labeled genomic DNA to control DNA microarrays. When used with sequence-mapped BACs (bacterial artificial chromosomes) a CGH provides a high-throughput whole genome approach to identifying genes amplified or deleted in cancers. Novel oncogenes and tumor suppressor genes may be identified using this approach.

A scoring system for liver impairment

A gene therapy technique in which a DNA:RNA oligonucleotide hybrid is introduced into a cell, allowing DNA repair mechanisms to introduce a (corrective) change in the targeted gene.

A chemical means of packaging foreign DNA in nanospheres that can be introduced into cells. Chitosan-DNA complexes have been tested in factor IX deficiency in animals.

This separation method depends on using a molecule that can preferentially bind to a protein of interest. Typical methodologies include using lectins (such as wheat germ or concanavalin A) to bind glycoproteins or using covalently coupled monoclonal antibodies to bind specific protein ligands.

This technique is designed to separate proteins based on their molecular weight. It is dependent on the exclusion of proteins from a matrix of specific size. Proteins that are too large to fit into the matrix of the gel bed run to the bottom of the column more quickly than smaller proteins, which are included in the volume of the matrix. Therefore, using appropriate size markers, the approximate molecular weight of a given protein can be determined and it can be separated from proteins of dissimilar size. Typical separation media for gel filtration chromatography include Sephadex and Ultragel.

This separation methodology depends on the preferential binding of positively charged proteins to a matrix containing negatively charged groups or a negatively charged protein binding to a matrix containing positively charged groups. Increases in the buffer concentration of sodium chloride are then used to break the ionic interaction between protein and matrix and elute off bound proteins. Examples of such separation media include DEAE and CM cellulose.

A general methodology to improve the separation of complex protein mixtures. The types of HPLC columns available are the same as for conventional chromatography, such as those based on size exclusion, hydrophobicity, and ionic interaction, but the improved flow rates resulting from the high pressure system provide enhanced separation capacity and improved speed.

This methodology separates proteins based on their hydrophobicity. Proteins preferentially bind to the matrix based on the strength of this interaction; proteins are then eluted off using solvents of increasing hydrophobicity. Separation media include phenyl-sepharose and octyl-sepharose.

These are regions of a gene either upstream, within, or downstream of the coding sequence that contain sites to which transcriptionally important proteins may bind. Sequences that contain 5 to 25 nucleotides are present in a typical cis-acting element.

This proto-oncogene encodes an ~45 kDa transcription factor that is a member of the AP1 family of transcriptional proteins. c-jun function depends on dimerization through its leucine zipper motif. Although c-jun-c-jun homodimers do form, they do so with low affinity and are not thought to be critical in gene transcription. Rather, a second partner, usually c-fos, generates the transcriptionally active heterodimer.

This gene encodes a transcription factor. It is expressed primarily in immature hematopoietic cells and declines as cells differentiate. Forced expression of c-myb blocks hematopoietic differentiation. Clinically, high levels of myb are noted in acute leukemia, and such patients are less likely to enter remission or tend to have a short remission duration.

This proto-oncogene plays a critical role in hematopoietic cell proliferation. Like the leucine zipper proteins, it too functions as a heterodimer. One of its partners is termed Max. The myc-related protein, Mad, also dimerizes with Max; the myc/Max complex stimulates proliferation, the Mad/Max complex inhibits myc-function. The importance of dysregulated myc function can be seen in Burkitt lymphoma in which a t(8;14) brings myc, on chromosome 8, into juxtaposition with the immunoglobulin locus on chromosome 14. Such upregulation of myc in a B lymphocyte setting results in a proliferative advantage and represents one important step in the genesis of this lymphoma. Myc has both leucine zipper and helix-loop-helix domains.

Three successive nucleotides on an mRNA that encode a specific amino acid in the polypeptide. Sixty-one codons encode the 20 amino acids, leading to codon redundancy. Three different codons signal termination of polypeptide synthesis.

Mutant or wild type-specific oligonucleotide primers are used in a PCR reaction with genomic DNA. The primers and stringency of PCR are chosen so that single-based mismatches between genomic DNA and PCR primer fail to yield an amplified product. Thus, the PCR detection of a locus-specific product allows the genotyping of the individual. An advancement over this technique is the color complementation assay, in which the wild type and mutation-specific PCR primers are labeled with different color fluorescent tags, and both are used in a PCR reaction with genomic DNA. When highly stringent conditions are met, the fluorescent colors of the resultant PCR product indicate whether wild type, mutant, or both specific alleles were present in the original DNA sample.

The jargon term used to describe the assembly of clones necessary to include all of the DNA in a specific stretch of chromosome. Such maps are usually assembled from overlapping YAC (yeast artificial chromosome) or BAC (bacterial artificial chromosome) clones. Once the “genome project” is complete, it will consist of 24 (very large) contigs (22 autosomal, an X and a Y).

By combining the elements of phage and plasmids, vectors can be constructed that carry up to 45 kb of foreign DNA.

This under-represented (i.e., < 1/16 frequency) dinucleotide pair is a “hotspot” for point mutation. CpG dinucleotides are often methylated on cytosine. Should Me-C undergo spontaneous deamination, uracil arises, which is then repaired by cellular surveillance mechanisms and altered to thymidine. The net result is a C to T mutation.

A group of proteins that vary in expression throughout the cell cycle. Once a threshold level is attained, interaction with specific cellular kinases results in phosphorylation of critical components of the mitotic machinery. Several classes of cyclins (A through E) exist that regulate different aspects of the cell cycle (G₀, G₁, S, G₂, M). Altered expression of some cyclins is associated with hematologic malignancy, e.g., t(11;14) in mantle cell lymphoma leads to overexpression of cyclin D₁, a G₁ phase cyclin.

A transfection method that depends on the formation of a complex between the insoluble positively charged dextran and the DNA to be transfected. Like calcium phosphate, this method is highly successful with many cell types.

Located on chromosome 6, DEK is involved in t(6:9) of AML. This translocation is usually seen in young patients and carries a poor prognosis. Its normal function is unknown, but DEK localizes to the nucleus.

This method relies on the random incorporation of dideoxynucleotides into a growing enzyme-catalyzed DNA chain. As no 3′ hydroxyl group is present on the ddN, chain synthesis halts following its incorporation into the chain. If ³²P or ³⁵S nucleotides are also incorporated into the reaction, a family of DNA fragments will be generated that can be visualized on a polyacrylamide gel. This method is presently the most commonly used method to determine the sequence of DNA.

To improve efficiency when screening functional expression libraries, many investigators construct cDNA libraries in which the proper coding orientation of the cDNA is maintained in the library. In conventional library preparation, the 5′ and 3′ ends of the DNA are identical; thus, cDNA can be inserted into the cloning vector in either orientation. If screening is dependent on the production of a functional protein, one-half of the library will be useless, as those cDNA inserted in an inverse orientation will not produce functional protein. Directional cloning is dependent on producing sticky ends that differ on the 5′ and 3′ termini. The cloning vector has the appropriate pair of complementary cloning sites.

The polymer constructed of successive nucleotides linked by phosphodiester bonds. Some 3 × 10⁹ nucleotides are contained in the human haploid genome. During interphase, DNA exists in a nucleoprotein complex containing roughly equal amounts of histones and DNA, which interacts with nuclear matrix proteins. This complex is folded into a basic structure termed a nucleosome containing approximately 150 base pairs. From this highly ordered structure, DNA replication requires a complex process of nicking, unfolding, replication, and splicing. In contrast, gene transcription requires nucleosomal reorganization such that sites critical for the binding of transcriptional machinery reside at internucleosomal junctions.

These enzymes are normally part of a bacterial host defense against invasion by foreign DNA. The enzyme normally methylates endogenous (host) DNA and thereby renders it resistant to a series of endogenous restriction endonucleases. In recombinant DNA work, methylation finds use in cDNA cloning to prevent subsequent digestion by the analogous restriction endonuclease.

Multiple (presently up to tens of thousands) gene fragments or oligonucleotides representing distinct genes spotted onto a solid support. Theoretically, microarrays could be used to determine the totality of the genome expressed in a given cell under specific growth conditions, if the entire genome were present on the microarray. At present, gene chips are available that represent about 1/3 of the human genome. The microarray is hybridized with a labeled probe (either radioactive or fluoresceinated) representing all the mRNA species in a given cell grown under a certain condition. By comparing the hybridization patterns produced by probes produced from cells under two different growth conditions, one can determine which genes are increased and which are decreased in response to the growth stimulus. In a similar way, comparison of the expression profiles of a malignant cell type and its normal counterpart, potentially allows one to determine the genes responsible for transformation.

An enzyme that synthesizes DNA from a DNA template. The intact enzyme purified from bacteria (termed the holoenzyme) has both synthetic and editing functions. The editing function results from nuclease activity. A modified DNA polymerase widely used in molecular biology studies is the Klenow fragment, a modified version of bacterial DNA polymerase that has been modified so that only the polymerase function remains; the 5′→3′ exonuclease activity has been eliminated.

This technique depends on the ability of protein specifically bound to DNA to protect the DNA from digestion by the endonuclease DNAse I. ³²P-labeled DNA is mixed with nuclear proteins, which potentially contain specific DNA-binding proteins, and the reaction is then subjected to limited DNAse digestion. If a given site of DNA is free of protein, it will be cleaved by the DNAse. In contrast, regions of DNAse specifically bound by proteins (transcription factors or enhancers) will be protected from digestion. The resultant mixture of DNA fragments from control and protein-containing reactions are then separated on a polyacrylamide gel. As the site of ³²P labeling of the original DNA fragment is known, sites that were protected from DNAse digestion will be represented on the gel as regions devoid of that length fragment. Therefore, in comparison to naked DNA, regions that bind specific proteins will be represented as a “footprint.”

This technique is designed to uncover regions of DNA that are in an “active” transcriptional state. It depends on the hypersensitivity of such sites (because of the lack of the highly compact nucleosome structure) to limited digestion with DNAse. Intact nuclei are subjected to limited DNAse digestion. The resultant large DNA fragments are then extracted, electrophoretically separated, and hybridized with a ³²P-labeled probe from a known site within the gene of interest. If, for example, the probe were located at the site of transcription initiation, and should DNA fragments of 2 kb and 5 kb be detected with this probe, hypersensitive sites would thereby be mapped to 2 kb and 5 kb upstream of the start of transcription initiation. By extrapolation, these sites would then be assumed to be important in the transcriptional regulation of the gene of interest, especially if such a footprint were only detected in cells that express that gene.

The only viral protein consistently expressed in EBV-associated malignancies. EBNA-1 binds in a site-specific manner to the viral DNA and is essential for viral replication, as well as for maintaining the EBV genome as an extrachromosomal episome within infected cells.

Many retroviruses are host cell specific, i.e., they will only infect cells from a specific species. An example is the widely used Maloney virus, and the basis for its specificity lies in the species-specific expression of the viral cell surface receptor.

Murine retroviruses that contain coat proteins that can only bind to murine cellular receptors.

When cells are suspended in buffer between two electrodes, discharge of an electrical impulse momentarily creates pores in the cell membrane. During this time, DNA in solution is free to diffuse into the cells. This method is highly successful in transfecting a large number of cell types, including cells previously thought to be difficult to transfect with other methods, such as endothelial cells and fibroblasts.

An enzyme that digests nucleic acids at specific sequences within the DNA.

An enhancer is a segment of DNA that lies either upstream, within, or downstream of a structural gene that serves to increase transcription initiation from that gene. A classical enhancer element can operate in either orientation and can operate up to 50 kb or more from the gene of interest. Enhancers are cis-acting in that they must lie on the same chromatin strand as the structural gene undergoing transcription. These cis-acting sequences function by binding specific proteins, which then interact with the RNA polymerase complex.

Refers to exogenous DNA that remains free in the target cell without being taken into the host genome.

A gene located on chromosome 8, ETO is involved in t(8:21) of AML type M2. It encodes the human homologue of the Drosophila Nervy protein.

More commonly known as the TEL oncogene (see Tel).

These are the regions of the primary RNA transcript that encode for protein. Following splicing, the exons are joined to form the mature mRNA species.

An enzyme that digests nucleic acids starting from the 5′ or 3′ terminus and extending inward.

A plasmid that contains all of the elements necessary to express an inserted cDNA in the host of interest. For a mammalian cell host, such a vector typically contains a powerful promoter coupled to an enhancer, a cloning site, and a polyadenylation signal. In addition, several expression vectors also contain a selectable marker gene such as DHFR or NeoR, which aids in the generation of stable cell lines. The plasmid also requires a bacterial origin of replication and an antibiotic resistance gene (AmpR) to allow propagation and expansion in a bacterial host.

Adds 15 carbon farnesyl groups to CAAX motifs, such as one present in ras, allowing their insertion into cellular membranes.

A transmembrane glycoprotein expressed on a wide variety of primitive and mature hematopoietic cells that, upon binding to its natural ligand, triggers programmed cell death.

A 40 kd type II membrane protein that induces apoptosis in cells expressing Fas (CD95). FasL is one of the major effectors of CD8⁺ CTL and NK activity. Loss of FasL leads to lymphoproliferative and autoimmune disease.

The retroviral enzyme reverse transcriptase is used along with an antisense primer to produce a complementary DNA strand of mRNA extracted from a cellular source known to express the gene of interest. Two types of primers are used, either oligo dT, in which the poly A tail begins the cDNA synthesis, or random primers, in which a whole range of start sites will be used.

A general method to assign chromosomal location, gene copy number (both increased and decreased), or chromosomal rearrangements. Biotin-containing nucleotides are incorporated into specific cDNA probes by nick-translation. Alternatively, digoxigenin or fluorescent dyes can be incorporated by enzymatic or chemical methods. The probes are then hybridized with solubilized, fixed metaphase cells, and the copy number of specific chromosomes or genes are determined by counterstaining with fluorescein isothiocyanate (FITC)-labeled avidin or other detector reagents. The number and location of detected fluorescent spots correlates with gene copy number and chromosomal location. The method also allows chromosomal analysis in interphase cells, allowing extension to conditions of low cell proliferation.

Simultaneous determination of the expression level of hundreds to thousands of genes by hybridization of RNA to cDNA or oligonucleotide arrays.

Specific genes in the mammalian genome can be targeted for interruption or correction based on the technique of homologous recombination. By generating DNA constructs that contain an interrupted gene of interest, or a corrected gene, in the setting of adequate flanking sequences to allow for targeting to the genetic locus of interest, the endogenous gene can be replaced or corrected. The methods involve introduction of the gene into an embryonic stem (ES) cell line, selection for subclones of cells that have had successful homologous recombination events, and then introduction of the ES subclone into the blastocyst of a developing embryo. A chimeric animal results, and should the newly introduced gene become part of the germline, it can be bred to the homozygous state. Using these techniques, investigators can determine whether a single genetic locus is responsible for a given disease, determine the significance of specific cytokines or growth factors, and generate model systems useful for the investigation of human disease. In a variant of this technique, gene knock-in experiments use the same targeting strategy, but rather than simply obliterating function of the targeted gene, the knock-in is designed to replace the locus with a specific mutation of interest.

A procedure for grouping genes with similar patterns of expression. Resulting clusters often consist of genes with related functions.

A transport mechanism contributing to drug resistance of cancer cells.

A prognostic system for chronic myelogenous leukemia.

This family of transcriptionally active proteins depends on the helix-turn-helix motif for dimerization. Examples include the homeodomain genes such as the Hox family.

A computational method that groups genes into clusters, and then groups the clusters into increasingly higher order collections. Ultimately, a dendrogram, or “tree,” denoting relationships between the groups emerges.

Transcription factors which were first identified as master switch transcriptional regulators of early development. Hox genes control body segmentation and more recently have been found to play a role in regulating hematopoietic stem cell survival and proliferation.

When exogenous DNA is introduced into a cell, it can be incorporated into the genome either randomly or at a specific locus. By incorporating sequences that normally flank the desired locus, a manipulated gene can be specifically (albeit rarely) introduced into the genome. Selection for this unlikely event can be enhanced by introduction of the herpes thymidine kinase (TK) gene into the original targeting construct. Should the construct be randomly incorporated into the genome, the TK gene will also be introduced, rendering the cell sensitive to gancyclovir. If homologous recombination occurs, the TK gene will be eliminated, as there are no homologous sequences at the specific genetic locus of interest and the resultant cell will be resistant to the antibiotic.

The antigenic specificity defined by the unique sequence (idiotope) of the antigen combining site on an immunoglobulin molecule.

Immunoglobulin variable region gene sequences are further diversified in mature B cells during clonal expansion that occurs following antigen stimulation. Mutations clustered within V regions typically involve nucleotide substitution, and less frequently small deletions or insertions. This event usually follows immunoglobulin class switching.

This technique is designed to detect specific RNA present in histological samples. Tissue is prepared with particular care not to degrade RNA. The cells are fixed on a microscope slide, allowed to hybridize to probe, and then washed and overlaid with photographic emulsion. Following exposure for one to four weeks, the emulsion is developed and silver grains overlying cells that contain specific RNA are detected. The most useful probes for this purpose are metabolically ³⁵S-labeled riboprobes generated by in vitro transcription of a cDNA using viral RNA polymerase. These probes give the lowest background and are preferable to using terminal deoxynucleotidyl transferase or alternative methods using ³²P as an isotope.

The ATG triplet is used to begin polypeptide synthesis. This is usually the first ATG codon, located approximately 30 nucleotides downstream of the site of transcription initiation (cap site). However, the context in which the ATG resides is also important (see KOZAK sequence).

This multi-protein complex forms at the site of transcription initiation and is composed of RNA polymerase, a series of ubiquitous transcription factors (TF II family), and specific enhancers and/or silencers. The proteins are brought together by the looping of DNA strands so that protein binding sites, which may range up to tens of kb apart, can be brought into close juxtaposition. Specific protein-protein interactions then allow assembly of the complex.

The mechanisms by which infection of a cell by one virus excludes infection by others. Interference is often due to the cellular production of coat proteins, which bind to and block the cells’ remaining viral receptors.

Regions of the primary RNA transcript that are eliminated during splicing. Their precise function is uncertain. However, several transcriptional regulatory regions have been mapped to introns, and they are postulated to play an important role in the generation of genetic diversity (exon shuffling mechanism).

A transcription factor that activates the expression of interferon α and β and maps to chromosome 5q31.1. As it is thought to act as a tumor suppressor gene, its role in the pathologic consequences of the 5q– syndrome is under active investigation.

A gene that binds to a promoter element shared by interferon α and β and many interferon-inducible genes. Unlike IRF-1, which stimulates such genes, IRF-2 represses transcription at the site. It is felt that the ratio of IRF-1 to IRF-2 might be a critical event in the regulation of cellular proliferation.

Restriction endonucleases that contain an identical recognition site but are derived from different species of bacteria (and hence have different names).

Class of immunoglobulin (IgG, IgM, IgA, etc.).

An inhibitor of NF-κB

The Ki-67 protein is a proliferation antigen, which is present in G₁, S, G₂ and M phases of the cell cycle. Quiescent or resting cells in the G₀ phase of the cell cycle do not express the Ki-67 antigen.

These enzymes transfer the g-phosphate group from ATP to the 5′ hydroxyl group of a nucleic acid chain.

A gene that encodes c-kit, a transmembrane receptor in the PDGF family that has tyrosine kinase activity and plays a role in hematopoiesis, gametogenesis and melanogenesis.

This five-nucleotide sequence resides just prior to the initiation codon and is thought to represent a ribosomal-binding site. The most consistent position is located three nucleotides upstream from the initiation ATG and is almost always an adenine nucleotide. When multiple potential initiation codons are present in an open reading frame, the ATG codon, which contains a strong consensus KOZAK sequence, is likely the true initiation codon.

Cis-acting sites are occasionally organized into a region removed from the structural gene(s) they control. Such locus control regions (LCRs) are best described for the β globin and α globin loci. First recognized by virtue of clustering of multiple DNAse hypersensitive sites, the β globin LCR is required for high-level expression from all of the genes and appears to be critical for their stage-specific developmental pattern of expression.

A family of DNA-binding proteins that dimerize by virtue of an alpha helical region that contains leucine at every seventh position. Because 3.4 amino acids reside in each turn of an alpha helix, the occurrence of leucine at every seventh position results in a strip of highly hydrophobic residues on one surface of the alpha helix. Such a domain on one polypeptide can intercalate with a similar domain on a second polypeptide, resulting in the formation of a stable homodimer or heterodimer. Examples of the leucine zipper family include the proto-oncogenes c-jun and c-fos.

Three major methods are available for screening to obtain a cDNA of interest. The classic technique utilizes DNA probes (such as oligonucleotides or intact cDNA from a homologous gene) to screen cDNA libraries. An oligonucleotide probe is usually derived from a reverse translation of known protein sequence. By expressing cDNA as a fusion protein with β-galactosidase, various antisera can be used to screen for fusion proteins encoded by the cDNA of interest. Finally, cDNA libraries may be constructed in cloning vectors that allow for expression of the cDNA insert in Escherichia coli or a mammalian cell host. If a highly sensitive assay for the desired protein’s function can be developed, pools of cDNA clones can be expressed and then assayed together; a positive assay from a pool would allow one to subdivide into smaller pools and eventually isolate a single clone.

These enzymes utilize the γ-phosphate group of ATP for energy to form a phosphodiester linkage between two pieces of DNA. The nucleotide contributing the 5′ hydroxyl group to the linkage must contain a phosphate, which is then linked to the 3′ hydroxyl group of the growing chain.

To efficiently insert the cDNA library into a cloning vector, synthetic duplex oligonucleotides that contain a restriction endonuclease site are attached to the blunted ends of the cDNA. A restriction endonuclease is chosen that rarely cuts DNA (such as the 8 base pair recognition sequence for Not I, or if a more common restriction site is used such as Eco RI, the cDNA should first be methylated in order to prevent subsequent cDNA digestion with the enzyme) and is used to generate “sticky ends” on the cDNA.

By encapsulating the DNA to be transfected in an artificial lipid carrier, foreign DNA can be introduced into the cell. This method, like electroporation, has been successful in transfecting cells previously thought difficult to manipulate.

This segment of a retroviral genome carries the genetic information for both transcription of downstream viral structural genes and the mechanisms of viral replication. It is often used in retroviral applications to drive the exogenous therapeutic gene as it carries a powerful (but non-tissue specific) promoter.

A stem-like cell that can be assayed in vitro by its ability to give rise to CFU-GM over many weeks in culture.

This gene, which is the most recent reporter gene to be used, has gained increasing acceptance because of its ease of assay and extreme sensitivity. The assay is based on the ability of the protein to undergo chemiluminescence and transmit light, detected with a luminometer.

These enzymes transfer phosphate groups from ATP to tyrosine, threonine, or serine residues of proteins. These enzymes are among the most important signaling molecules present in mammalian cell biology.

These polypeptide products are thought to regulate a cassette of genes that induce a cell to undergo a new program of differentiation. An example of such a system is Myo D, activation of which is thought to lead to differentiation along the muscle cell lineage.

A method to determine the sequence of a stretch of DNA based on its differential cleavage pattern in the presence of different chemical exposures. A nucleic acid chain can be cleaved following G, A, C, or C and T by exposure of ³²P-labeled DNA to neutral dimethylsulfate, dimethylsulfate-acid, hydrazine-NaCl-piperidine or hydrazine-piperidine alone, respectively.

A transcriptional regulator that plays a major role in regulating expression of p53.

A quantitative method of combining the results of independent studies (usually drawn from the published literature) and synthesizing summaries and conclusions that may be used to evaluate therapeutic effectiveness.

Monoclonal antibody developed against the Ki-67 antigen.

Mutation of the mRNA sequence to generate an altered codon, which results in an amino acid change.

A gene, located on chromosome 11, MLL (for mixed lineage leukemia) is frequently altered in ALL, primary AML, and especially in AML secondary to the use of topoisomerase II inhibitors. MLL is homologous to the trithorax gene of Drosophila and displays many features of a transcription factor and of a DNA methyl transferase.

Like DNAse footprinting, this technique is used to determine whether a fragment of DNA binds specific proteins. ³²P-labeled DNA (either duplex oligonucleotides or small restriction fragments) are incubated with nuclear protein extracts and subjected to native acrylamide gel electrophoresis. When specific DNA-binding proteins that recognize the oligonucleotide or restriction fragment probe are present in the nuclear extracts, a DNA-protein complex will be formed and its migration through the native gel will be retarded compared to the unbound DNA. Hence, the labeled band will be shifted to a more slowly migrating position. The specificity of the reaction can be demonstrated by also incubating, in separate reactions, competitor DNA that contains the presumed binding site or irrelevant DNA sequence.

Several methods are now available to introduce specific mutations into a cDNA sequence of interest. Most are based on designing an oligonucleotide that contains the desired mutation in the context of normal sequence. This oligonucleotide is then incorporated into the cDNA using DNA polymerase, either using a single-stranded DNA template (phage M13) or in a PCR format to produce a heteroduplex DNA containing both wild type and mutant sequences. Using M13, recombinant phage are then produced and mutant cDNA are screened for on the basis of the difference in wild type and mutant sequences; using the PCR format, the exponential amplification of the mutant sequence results in its overwhelming numerical advantage over wild type sequence, resulting in nearly all clones containing mutant sequence. Both of these methods require that the entire cDNA insert synthesized in vitro be sequenced in its entirety to guarantee the fidelity of mutagenesis and synthesis of the remaining wild type sequences.

By using an independent set of PCR primers located within the sequence amplified by the primary set, the specificity of a PCR reaction can be greatly enhanced.

The gene responsible for neurofibromatosis. The normal protein functions to negatively regulate ras proteins, key intermediates in cytokine-induced cellular proliferation.

A histone-like CCAAT-binding protein that cooperates with E2 factors (E2Fα) to regulate transcription of many cell cycle genes. NF-Y is the prototype of a constitutive, ubiquitous factor that pre-sets the promoter architecture for other regulatory proteins to access it.

Is a transcription factor that plays an important role in regulating immune response, embryonic and cell lineage development, apoptosis, cell cycle progression, tumor formation, oncogenesis and viral replication.

This technique is used to label cDNA to high specific activity for the purpose of probing Southern and Northern blots and screening cDNA libraries. The cDNA fragment is first nicked with a limiting concentration of DNAse, then DNA polymerase is used to both digest and fill in the resulting gaps with labeled nucleotides.

Non-obese diabetic mouse bred for homozygosity of severe combined immunodeficiency disease trait. Widely employed for in vivo stem cell assays, and for growth of xenogenic tumors.

In vivo assay for stem cells present within a defined population of peripheral blood or bone marrow cells.

A mutation that results in the generation of a premature termination codon and hence creates a truncated polypeptide.

Nonviral methods include polylysine-ligand DNA complexes, where the ligand (e.g., transferrin) allows access to the cell through normal receptor-mediated uptake, and phospholipid vesicles. Both methods suffer from not providing a mechanism for genomic integration, precluding long-term expression.

This modification of a Southern blot is used to detect specific RNA. The sample to be size-fractionated in this case is RNA and, with the exception of denaturation conditions (alkali treatment of the Southern blot versus formamide/formaldehyde treatment of the RNA sample for Northern blot), the techniques are essentially identical. The probe for Northern blotting must be antisense.

Member of a Type 1 transmembrane receptor family that shares structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like (EGF) repeats, and an intracellular domain consisting of multiple, different domain types. Notch family members play a role in a variety of developmental processes by controlling cell fate decisions.

When linear, the length of a specific chromosome is many orders of magnitude greater than the diameter of the nucleus. Therefore, a mechanism must exist for folding DNA into a compact form in the interphase nucleus. Nucleosomes are complex DNA protein polymers in which the protein acts as a scaffold around which DNA is folded. The mature chromosomal structure then appears as beads on a string; within each bead (nucleosome) are folded DNA and protein. Nucleosome structure is quite fluid, and internucleosomal stretches of DNA are thought to be sites that are important for active gene transcription.

The term given to any stretch of a chromosome that could encode a polypeptide sequence, i.e., the region between a methionine codon (ATG) that could serve to initiate protein translation, and the in-frame stop codon downstream of it. Several features of the ORF can be used to judge whether it actually encodes an expressed protein, including its length, the presence of a “Kozak” sequence upstream of the ATG (implying a ribosome might actually bind there and initiate protein translation), whether the ORF exists within the coding region of another gene, the presence of exon/intron boundary sequences and their splicing signals, and the presence of upstream sequences that could regulate expression of the putative gene.

Wild-type p53 is a sequence-specific DNA-binding nuclear protein that acts to induce gene expression. Overall, the program of p53-activated genes is associated with suppression of cell growth, consistent with our understanding of the action of anti-oncogenes. Mutations of p53 may not only inactivate its growth-suppression function, but can actually generate a genetically dominant, functional oncogene. Human tumors associated with p53 mutations include those of hematopoietic tissues (e.g., 20% of myelomas), bladder, liver, brain, breast, lung, and colon. It is likely the most frequently mutated gene in human cancer.

Form of signaling in which the target cell is close to the signal releasing cell.

A biosynthetic precursor of the ABH and P1 blood group glycosphingolipids and of one class of gangliosides

This technique finds use in several arenas of recombinant DNA technology. It is based on the ability of sense and antisense DNA primers to hybridize to a cDNA of interest. Following extension from the primers on the cDNA template by DNA polymerase, the reaction is heat-denatured and allowed to anneal with the primers once again. Another round of extension leads to a multiplicative increase in DNA products. Therefore, a minute amount of cDNA can be efficiently amplified in an exponential fashion to result in greatly increased quantities of cDNA. By including critical controls, the technique can be made quantitative.

Fused with TEL in CMML (see ETV6).

A virus of bacteria, phage such as lambda have been used to introduce foreign DNA into bacteria. Because of its infectious nature, the transfection (introduction) efficiency into the bacterial host is usually two orders of magnitude greater for phage over that of plasmids.

Autonomously replicating circular DNA that are passed epigenetically between bacteria or yeast. In order to propagate, plasmids must contain an origin of replication. Naturally occurring plasmids transfer genetic information between hosts; of these, the genes encoding resistance to a number of antibiotics are the most important clinically. The essential components of plasmids are used by investigators to introduce genes into bacteria and yeast and to generate large amounts of DNA for manipulation.

Following transcription of a gene, a specific signal near the 3′ end of the primary transcript (AATAAA) signals that a polyadenine tail be added to the newly formed transcript. The tail may be up to several hundred nucleotides long. The precise function of the poly A tail is uncertain but it seems to play a role in stability of the mRNA and perhaps in its metabolism through the nuclear membrane to the ribosome.

Refers to the observation that the site of vector integration into the genome often results in variable levels of gene expression.

Mechanisms of gene regulation that do not involve transcriptional enhancement or silencing and include altering the rate of mRNA degradation, the efficiency of translation or post-translational modification, or transportation of the polypeptide out of the cell.

Prediction analysis of microarrays (PAM) A statistical method that identifies groups of genes whose expression best characterizes a predefined class, and then uses these genes to predict their likelihood of expression in new samples.

This term is applied to the assembly of a polypeptide sequence from mRNA.

The general term used in the study of the display of all proteins present in cells under defined conditions. By deciphering which proteins are differentially displayed in tumor cells compared to their normal counterparts, or in cells stimulated to grow in comparison to their quiescent state, one can determine the proteins that are responsible for the cellular phenotype. In essence, proteomics is to proteins what genomics is to genes.

A large multiprotein complex designed to digest proteins that have been targeted for destruction, usually based on the presence of multiple sites of ubiquination. The proteosome is critical for many cellular processes, including cell growth, where it eliminates a series of brakes on cell cycle progression, or eliminates growth factor receptors following their internalization following ligand binding.

Selectins are a family of cell adhesion molecules consisting of a lectin-like domain, an epidermal growth factor-like domain, and a variable number of domains that encode proteins homologous to complement-binding proteins. Selectins mediate the binding of leukocytes to the vascular endothelium. P-selectin mediates the adhesion of neutrophils and monocytes to activated platelets and endothelial cells.

These take advantage of the powerful expression levels obtainable by murine retroviral backbones, yet are packaged in an envelope that allows docking and uptake by human target cells. An example is the popular MFG vector that utilizes a murine leukemia retroviral backbone and an amphotropic packaging cell line to produce infectious particles.

This gene encodes SHP-2, a non-receptor Src-homology 2 domain-containing protein tyrosine phosphatase. SHP-2 plays an important role in intracellular signaling elicited by growth factors, hormones, and cytokines, and it is required during development and hematopoiesis. Gain of function mutations in PTPN11 are observed in Noonan syndrome and related development disorders, as well as in myeloid malignancies.

Required for generation of antibody diversity. May play a role in lymphomagenesis.

This technique is also used to produce labeled cDNA probes and is dependent on using random 6- to 10-base oligonucleotides to sit down on a single-stranded cDNA and then using DNA polymerase to synthesize the complementary strand using labeled nucleotides. This technique usually produces more favorable results than nick-translation.

The retinoic acid receptor is a member of the steroid hormone group of transcriptionally active proteins and contains a steroid hormone-binding domain, a zinc finger DNA-binding domain, and a transcriptional activation domain. RAR is located at the t(15;17) breakpoint present in the majority of cases of acute promyelocytic leukemia. Its fusion partner in the translocation is termed pml. Normally, RAR forms heterodimers with members of the RXR family of transcription factors.

This gene encodes a critical signaling intermediate involved in the response to multiple growth factors. There are several related proteins (Ha-ras, Ki-ras, N-ras). N-ras and K-ras are mutated in many cancers, including 45% of myelomas and > 50% of CMML cases. Constitutive activation of ras can mimic chronic stimulation by the corresponding lineage-specific growth factor.

The prototypical tumor suppressor gene Rb behaves in a genetically recessive fashion. Elimination or inactivation of both Rb gene copies is required for manifestation of the tumorigenic phenotype, first recognized in children with retinoblastoma. Such children inherit only a single functional copy; subsequent mutagenic inactivation of the remaining allele results in tumor susceptibility. Rb acts to sequester a group of transcription factors, termed E2F, which regulate genes critical for DNA synthesis. Alterations of Rb alleles are found in approximately 30% of human acute leukemias.

A molecular method to amplify genes that are expressed in an RNA sample of interest, that are not present, or present at very reduced levels, in a comparison RNA sample (e.g., cytokine induced and control cells). The method relies on RT-PCR amplification of the RNA that does not contain the gene(s) of interest to produce a “driver” cDNA, and RT-PCR to produce “tester” cDNA from the RNA population in which you hope to find new genes. After ligation of different oligonucleotides to the ends of each population, both are denatured and an excess of the driver is hybridized to the tester and PCR performed with primers that will amplify only sequences present in the tester that are not in the driver, thereby “removing” cDNA common to both populations. The resultant cDNA are enriched in uniquely expressed genes.

During PCR, a fluorogenic probe, consisting of an oligodeoxynucleotide with both reporter and quencher dyes attached, anneals between the two standard PCR primers. When the probe is cleaved during the next PCR cycle, the reporter is separated from the quencher so that the fluorescence at the end of PCR is a direct measure of the amplicons generated throughout the reaction. Such a system is amenable to automation and gives precise quantitative information.

In order to determine how a gene promoter or enhancer works in vitro, that genetic element is often linked to a gene for which a simple assay is readily available and whose regulation is not affected by post-transcriptional processes. Such reporter genes include chloramphenicol acetyl transferase, b galactosidase, and firefly luciferase. The first is the most commonly used reporter; however, more recent studies have emphasized the use of the latter two reporters, as these are more sensitive to minimal changes in promoter or enhancer activity.

These enzymes are among the most useful in recombinant DNA technology, capable of introducing a single cleavage site into a nucleic acid. The site of cleavage is dependent on sequence; recognition sites contain from 4 to 10 specific nucleotides. The resultant digested ends of the nucleic acid chain may either be blunt or contain a 5′ or 3′ overhang ranging from 1 to 8 nucleotides.

This automated variant of allele-specific hybridization couples unlabeled synthetic oligonucleotides specific for a wild type or mutant sequence to a solid support that is then allowed to bind genomic sequences of the locus of interest, which have been amplified by PCR. The use of highly stringent conditions of hybridization allows differential binding of the amplified DNA to the wild type or mutant specific oligonucleotide and thereby allows determination of genotype.

Often, large families of homologous proteins exist and multiple previously unknown members of the family can be obtained by screening cDNA libraries under low stringency using cDNA or oligonucleotide probes from regions highly conserved amongst members of the family. In this case, genes are identified before their function is known, a situation referred to as reverse genetics. Examples in hematology include identifying members of the tyrosine kinase family of receptor proteins using a probe derived from the conserved kinase domain of the cytoplasmic region of src or other tyrosine kinase proto-oncogenes, or the identification of transcription factors important in hematopoiesis using conserved motifs present in zinc finger or homeodomain proteins.

This enzyme, first purified from retrovirus-infected cells, produces a cDNA copy from an mRNA molecule if first provided with an antisense primer (oligo dT or a random primer). This enzyme is critical for converting mRNA into cDNA for purposes of cloning, PCR amplification, or the production of specific probes.

If a mutation of one allele of a genetic locus either generates or destroys a restriction endonuclease site, the heterogeneity present within or very close to a gene of interest can be used to track which allele an individual has inherited from each parent. When genomic DNA is digested with a restriction enzyme that recognizes a polymorphic site and then hybridized with a probe specific for the gene of interest, the allelic pattern can be compared to that of a similar assessment of both parents. The presence of multiple family members allows a complete genetic pedigree to be constructed. For example, globin gene mutations such as sickle hemoglobin can be analyzed. The β6 mutation in hemoglobin, which results in Hgb S, destroys an Mst II site. Therefore, a larger than normal DNA fragment is generated by digestion of genomic DNA with Mst II, which can be easily detected by Southern blot hybridization. In this specific case, the Mst II polymorphism is absolutely specific for the mutant gene and family studies are not necessary. If the RFLP had not been specific for the mutation, but only existed close to the specific disease-producing mutation, then family studies would have been required to determine which pattern (presence or absence of restriction site) tracks with the mutant (disease) allele.

These enzymes degrade RNA and exist as either exonucleases or endonucleases. The three most commonly used ribonucleases are termed RNase A, RNase T1, and RNAse H (which degrades duplex RNA or the RNA portion of DNA•RNA hybrids).

These labeled RNA molecules are produced by first cloning the cDNA of interest into a plasmid vector that contains promoters for viral RNA polymerases. Following cloning, the viral RNA polymerase is added, and labeled nucleotides are incorporated into the resulting RNA transcript. This molecule is then purified and used as a probe in hybridization studies. Many such cloning vectors (for example, pGEM) have different RNA polymerase promoters on either side of the cloning site, allowing the generation of both sense and antisense probes from the same construct.

Based on a catalytic RNA characterized by a hammerhead-like secondary structure. Introduction of specific sequences into its RNA recognition domain targets specific mRNA species for destruction. Ribozymes thus represent a tool to eliminate expression of specific genes, and are being tested in several hematological disease states, including neoplasia. A highly specific RNA sequence can generate secondary structure by virtue of intrachain base pairing. “Hairpin loops” and “hammer head” structures serve as examples of such phenomena. When the proper secondary structure forms, such RNA molecules can bind a second RNA molecule (e.g., an mRNA) at a specific location (dependent on an approximately 20-nucleotide recognition sequence) and cleave at a specific GUX triplet (where X = C, A, or U). These molecules will likely find widespread use as tools for specific gene regulation or as antiviral agents.

Three varieties of RNA are easily identified in the mammalian cell. Most abundant is ribosomal RNA (rRNA), which occurs in two sizes, 28S (approximately 4600 nucleotides) and 18S (approximately 1800 nucleotides); together they form the basic core of the eukaryotic ribosome. Messenger RNA (mRNA) is the term used to describe the mature form of the primary RNA transcript of the individual gene once it has been processed to eliminate introns and to add a polyadenylated tail. mRNA links the coding sequence present in the gene to the ribosome, where it is translated into a polypeptide sequence. Transfer RNA (tRNA) is the form of RNA used to shuttle successive amino acids to the growing polypeptide chain. A tRNA molecule contains an anticodon, a three-nucleotide sequence by which the tRNA molecule recognizes the codon contained in the mRNA template, and an adapter onto which the amino acid is attached.

This enzyme is used by mammalian cells to transcribe structural genes that result in mRNA. The enzyme interacts with a number of other proteins to initiate transcription, including a number of general factors, and tissue-specific and induction-specific enhancing proteins.

This enzyme is used by the cell to transcribe ribosomal RNA genes.

This assay is in many ways similar to the S₁ nuclease analysis. In this case, a ³⁵S- or ³²P-labeled antisense RNA probe is synthesized and hybridized with mRNA of interest. The duplex RNA is then subjected to digestion with RNAse A and T₁, both of which will cleave only single-stranded RNA. Following digestion, the remaining labeled RNA is size-fractionated, and the size of the protected RNA probe then gives an indication of the size of the mRNA present in the original sample. This assay can also be used to quantitate the amount of specific RNA in the original sample.

This technique allows the rapid amplification of cDNA starting with RNA. The first step of the reaction is to reverse-transcribe the RNA into a first strand cDNA copy using the enzyme reverse transcriptase. The primer for the reverse transcription can either be oligo dT, to hybridize to the polyadenylation tail, or the antisense primer that will be used in the subsequent PCR reaction. Following this first step, standard PCR is then performed to rapidly amplify large amounts of cDNA from the reverse transcribed RNA.

This technique is used to identify the start of RNA transcription. The DNAse enzyme S₁ cleaves only at sites of single-stranded DNA. Therefore, if ³²P-labeled DNA is hybridized with mRNA, the resulting heteroduplex can be digested with S₁, and the resulting DNA fragment will be of length equivalent to the site at which the piece of DNA begins through the mature 5′ end of the RNA.

A mouse strain suitable for transplantation of human hematopoietic cells and malignancies

This proto-oncogene, first identified in a stem cell leukemia at the site of t(1;14), is a member of the helix-loop-helix group of transcriptionally active proteins. The gene, also termed Tal 1, is expressed in erythroid and mast cell lineages but not in T cells. The association of t(1;14) with up to 25% of T cell ALL suggests that its ectopic expression is associated with transformation.

The enzyme DNA polymerase is used to generate the sense strand of cDNA. Priming of the second strand can occur spontaneously, as the antisense first cDNA strand can form a hairpin loop at its 3′ end bending back to prime second strand synthesis. Alternatively, a polynucleotide tail can be added to the first strand synthesis using terminal deoxynucleotide transferase, then second strand priming can occur using a synthetic oligonucleotide complementary to the TdT tail. Should the former technique be used, an extra step to nick the hairpin loop using the enzyme S₁ nuclease would be required prior to inserting the cDNA into its vector.

These elements are very similar to enhancers except that they have the function of binding proteins and inhibiting transcription.

A prognostic system for chronic myelogenous leukemia.

This technique is used to detect specific sequences within mixtures of DNA. DNA is size-fractionated by gel electrophoresis and then transferred by capillary action to nitrocellulose or another suitable synthetic membrane. Following blocking of nonspecific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with a cDNA or oligonucleotide probe representing the specific DNA sequence of interest. Should specific DNA be present on the blot, it will combine with the labeled probe and be detectable by autoradiography. By co-electrophoresing DNA fragments of known molecular weight, the size(s) of the hybridizing band(s) can then be determined. For gene rearrangement studies, Southern blotting is capable of detecting clonal populations that represent approximately 1% of the total cellular sample.

This technique is designed to detect specific DNA-binding proteins. Like the Western blot, proteins are size-fractionated and transferred to nitrocellulose. The probe in this case, however, is a double-stranded labeled DNA that contains a putative protein-binding site. Should the DNA probe hybridize to a specific protein on the blot, that protein can be subsequently identified by autoradiography. This technique often suffers from nonspecificity, so that a number of critical controls must be included in the experiment for the results to be considered rigorous.

The primary RNA transcript contains a number of sequences that are not part of the mature mRNA. These regions are called introns and are removed from the primary RNA transcript by a process termed splicing. A complex tertiary structure termed a lariat is formed and the intron sequence is eliminated bringing the coding sequences (exons) together. Specific sequences within the primary transcript dictate the sites of intron removal.

The purpose of generating a subtractive library is to enrich for cDNAs that are expressed under one condition but are not expressed under a second condition. This facilitates screening for the cDNA of interest because the complexity of the library is much reduced, requiring one to screen far fewer clones. At its extreme, investigators have used subtractive libraries to generate a very highly select group of clones (in the range of 100) and then have sequenced all of the resulting cDNA. The principle behind a subtractive library is the elimination of cDNA common to induced and control conditions. By eliminating such clones, only cDNA that are present under the induced conditions will remain in the library. Those techniques depend on the differential elimination of duplex mRNA/cDNA or cDNA/cDNA hybrids, which form between genes expressed under both conditions, leaving the single-stranded mRNA or cDNA of interest.

Supervised analysis An analysis of microarray expression profiling results that takes into account a set of external factors defined by the user.

Many genes have a sequence that includes this tetranucleotide close to the beginning of gene transcription. RNA polymerase binds to the sequence and begins transcription at the cap site, usually located approximately 30 nucleotides downstream.

A helix-loop-helix transcription factor fused to the PDGFβ-receptor in CMML t(5:12) and to other genes in AML or MDS. Like most other translocation oncogenes, the mechanism of leukemogenesis is unknown. More recently, a TEL/AML1 fusion gene representing a t(12;21) has been found in a large number of cases of childhood ALL. As the translocation is not detected by routine cytogenetics, molecular analysis (FISH, etc.) is required to identify this favorable chromosomal rearrangement.

A specialized DNA polymerase that protects the length of the terminal segment of a chromosome. Should the telomere become sufficiently shortened (by repeated rounds of cell division), the cell undergoes apoptosis. The holoenzyme contains both a polymerase and an RNA template; only the latter has been characterized, although the gene for the enzymatic activity has recently been cloned.

A repeating structure found at the end of chromosomes, serving to prevent recombination with free-ended DNA. Telomeres of sufficient length are required to maintain genetic integrity, and they are maintained by telomerase.

This lymphocyte-specific enzyme normally transfers available (random) nucleotides to the 3′ end of a growing nucleic acid chain. In recombinant DNA technology, these enzymes can be used to add a homogeneous tail to a piece of DNA, thereby allowing its specific recognition in PCR reactions or in cloning efforts.

The prototype polymerase, Taq, and newer versions such as Vent and Tth polymerase are derived from microorganisms that normally reside at high temperature. Consequently, their DNA polymerase enzymes are quite stable to heat denaturation, making them ideal enzymes for use in the polymerase chain reaction.

Induces cytolysis and cytostasis of many tumor cell lines in vitro. TNF-α also enhances phagocytosis and cytotoxicity in neutrophilic granulocytes and modulates the expression of many other proteins, including fos, myc, IL-1 and IL-6.

A homodimeric chromosomal unwinding enzyme that introduces a double-stranded nick in DNA, which allows the unwinding necessary to permit DNA replication, followed by religation. Inhibition of topoisomerases leads to blockade of cell division, the target of several chemotherapeutic agents (e.g., etoposide).

Kills activated lymphocytes when it binds to its receptor.

Specific proteins that bind to control elements of genes. Several families of transcription factors have been identified and include helix-loop-helix proteins, helix-turn-helix proteins, and leucine zipper proteins. Each protein includes several distinct domains such as activation and DNA-binding regions.

Transcription is the act of generating a primary RNA molecule from the double-stranded DNA gene. Regulation of gene expression is predominantly at the level of regulating the initiation and elongation of transcription. The enzyme RNA polymerase is the key feature of the system, which acts to generate the RNA copy of the gene in combination with a number of important proteins. There is usually a fixed start to transcription and a fixed ending.

Gene regulation is determined by the rate of transcriptional initiation. This usually results from alteration in the level of activity of transacting proteins, which, in turn, are regulated either by the amount of the transcriptionally active protein or by their state of activation.

The act of transferring a foreign gene into a host genome, usually via a retrovirus.

Introduction of foreign DNA by mechanical methods. Several methods are available for such transfections and include calcium/phosphate/DNA complexes, DEAE Dextran, electroporation, liposome, and retrovirus-mediated gene transfer.

By introducing an intact or manipulated gene into the germline of mice, the effects of promoter expression in specific cell lineages can be investigated. In contrast to highly artificial in vitro studies using reporter gene analysis, such transgenic animals provide an important in vivo model of gene function. The methods for production of transgenic mice have been extensively reviewed and are based on the microinjection of linear DNA into the pronucleus of a fertilized egg. Several types of experiments can be performed. First, the effect of aberrant expression of a gene can be investigated, as was recently performed by expressing GM-CSF in a wide variety of tissues. Second, the necessary elements for tissue- and developmental level-specific expression of a gene can be studied, as has been performed for the β-globin locus. Third, the tissue distribution of a specific gene can be determined by engineering a marker gene adjacent to a specific promoter. A specific example of this strategy employs a “suicide gene,” the herpes virus thymidine kinase (TK). When animals carrying such genes are exposed to gancyclovir, cells expressing the promoter of interest will express TK, be killed, and be readily detected.

Naturally occurring genetic elements that are naturally easily removed and inserted into the genome, allowing for the recombination of genetic segments, giving rise to genetic diversity. These same elements can be utilized for gene therapy.

The Trk proto-oncogene family contains four members, TrkA, TrkB, TrkC, and TrkE which are variably expressed throughout the central and peripheral nervous systems. Activated TrkA binds to nerve growth factor (NGF) and autophosphorylates on tyrosine residues (Tyr490, Tyr674, Tyr675, Tyr 751, and Tyr785) leading to activation of multiple downstream effector proteins.

A small molecular weight protein that can be coupled to lysine residues of proteins targeted for destruction. There are ubiquitin ligases and deubiquinases, which add or remove ubiquitin from proteins, usually in a fairly specific manner. Once polyubiquinated, proteins are subject to destruction by the proteosome.

An analysis of microarray expression profiling results that does not consider external factors concerning the samples.

Retroviral vectors are based on murine retroviruses. They can carry 6 to 7 kb of foreign DNA (promoter + cDNA) but suffer from the drawbacks of requiring the development of high titer packaging lines, requiring that target cells be dividing, and are subject to host cell down-modulation. Adenoviral vectors can be produced at high levels and do not require a dividing target cell, but they do not normally integrate, resulting in only transient expression. Adeno-associated viral vectors are defective parvoviruses that integrate into a nondividing host cell at a specific location (19q). Disadvantages are genetic instability, small range of insert size (2–4.5 kb), and thus far, only transient expression.

These enzymes are utilized in recombinant DNA technology to transfer phosphate groups (either unlabeled or ³²P-labeled) to oligonucleotides or DNA fragments. The most commonly used kinase is T4 polynucleotide kinase.

Rod-shaped bundles of microtubules seen by electron microscopy in vascular endothelial cells. Weibel-Palade bodies are the von Willebrand factor storage compartment of human umbilical vein endothelial cells.

Western blotting This technique is designed to detect specific protein present in a heterogenous sample. Proteins are denatured and size-fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose or other synthetic membranes, and then probed with an antibody to the protein of interest. The immune complexes present on the blot are then detected using a labeled second antibody (for example, a ¹²⁵I-labeled or biotinylated goat anti-rabbit IgG). As the original gel electrophoresis was done under denaturing and reducing conditions, the precise size of the target protein can be determined.

A family of highly conserved, secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis that are thought to control cell fate decisions during development. Wnt proteins constitute a large family of secreted molecules that are involved in intercellular signaling during development. The name derives from the first 2 members of the family to be discovered: int-1 (mouse) and wingless (Drosophila).

Several loci present on the X chromosome become highly methylated when inactive but remain unmethylated on the active X chromosome (Lyon hypothesis). Should a polymorphic site for a methylation-sensitive restriction endonuclease exist at such an X-linked locus, one can distinguish between the active and inactive X chromosome by the pattern of restriction endonuclease digestion of that gene. However, in order to be widely useful for determining clonality of hematopoietic cells, the allelic frequency must be close to equality. Several X-linked genes meet these criteria and include phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyltransferase (HPRT), the human androgen receptor gene (HUMARA), and the hyper-variable DXS255 locus. Both Southern blotting and PCR methods can be applied to this type of analysis.

A yeast artificial chromosome (YAC) utilizes centromeric and telomeric elements from yeast chromosomes to construct genetic elements that can be propagated in yeast and transferred into mammalian cells. Such vehicles allow the introduction of up to 200 kb or more of genetic material into the host cells. YACs are now being used to study the physiologic regulation of large genetic loci such as the b-globin region of chromosome 11.

A strategy designed to determine the binding partners for a protein of interest. The gene (or a fragment of the gene) representing a protein of interest (the “bait”) is fused in frame to DNA binding domain (DBD) of yeast transcription factor and then introduced into a yeast strain. A cDNA library is then constructed from the cells in which the bait is normally expressed, and fused in frame to the activation domain (AD) of the same yeast transcription factor. When the library is introduced into the yeast expressing the bait/DBD fusion, any yeast cell expressing a cDNA encoding a binding partner of the bait protein will have that cDNA/AD fusion protein bind to the bait/DBD fusion, bringing the AD and DBD together, thereby creating a fully functional transcription factor that now drives a reporter gene, allowing the yeast carrying such interacting proteins to be identified and the cDNA recovered.

A surface antigen on lymphocytes. Surface expression of ZAP 70 has been shown to be a prognostic marker in CLL.

This is a large family of transcriptionally active proteins that includes the steroid hormone receptors. The presence of conserved histidine and cysteine residues allows chelation of a zinc atom and results in the formation of a loop structure called the zinc finger domain.

An inactive precursor of an enzyme, particularly a proteolytic enzyme.