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CLIN. CHEM. 31/6, 804-811 Applications (1985) of Recombinant C Dan Sauls”2 and C. Thomas DNA to Pathologic Caskey’ Recombinant DNA techniques are contributing to the understanding of the pathogeneses of genetic, neoplastic, and viral diseases, and are used in the diagnosis of certain genetic and viral diseases. Such techniques will have tion in the future and will play an increasing laboratory. The technology wider applica- role in the clinical of this field rests upon the cleav- age of DNA by certain enzymes, restriction endonucleases, and upon the ability to locate specific sequences of nucleotides in a cleaved DNA sample by using known fragments of DNA labeled (“probes”) with radioisotopes or biotin. To produce useful probes, one “clones” multiple copies of the same DNA fragment in bacteria. The use of DNA probes in the clinical laboratory is valuable in antenatal diagnosis, genetic counseling, and post-natal diagnosis of genetic dis- eases, especially metabolism. genetic hematologic DNA material probes in clinical diseases and inborn errors of can also be used specimens. DNA probes fetal status cancer Lesch-Nyhan disease AdditIonal Keyphrases: to detect viral heritable disorders genetics viruses restriction endonucleases hemoglobin variants Huntington’s disease Duchenne’s muscular dystrophy Ieukemias Wilms’s tumor retinoblastoma . AIDS Recombinant industrial science. DNA research, technology basic . has significantly biological affected science, and medical science disciplines and field utilizes many basic promises significant advancements in laboratory diagnosis. It has already contributed significantly to our understanding of the pathogenesis of inherited and virally acquired diseases. We present some examples of current applications in this review, together with an appropriate terminology and illustrations of the technology. The Methods Principle of Hybridization DNA can be readily for use in molecular isolated from hybridization strong acids can destroy is stable to extraction its inherent procedures, various cellular Glossary such that adenine pairs with thymidine and quanine pairs with cytidine Vol. 31, No. 6, 1985 (or uracil). Linkage: The linear association of genes on the same DNA molecule, determined by the distance between two genes. The closer two genes are on a chromosome, the greater is the chance of their being inherited together; the further the linkage distance, the greater the likelihood they will be inherited independently. Oncogene: A gene involved in the process of neoplastic transformation. Viral DNA that replicates independently of the DNA of the host cell. The proteins resulting from plasmid DNA confer antibiotic resistance to host bacteria. Point mutation: A change in one nucleotide of a gene, in which one base replaces another. Promoter: A segment of DNA involved in binding the enzymes necessary to initiate transcription of DNA into RNA. Restriction endonuclease: An enzyme that can cleave doublestranded DNA at specific sequences of nucleotides, usually at sequences four to six bases long. Restriction fragment: A DNA fragment generated by the cleavage of a parent molecule by a restriction endonuclease. Transcription: The process that produces a complementary strand of RNA from DNA. Plasmid: Restriction Endonucleases Because DNA is a complex polymer of high molecular mass, its efficient fragmentation is required for detailed gene study. “Restriction endonucleases” are enzymes capable of cleaving double-stranded DNA at specific sequences of nucleotides, four to six bases long (Figure 1) (1). Cleavage of the large DNA molecule reproducibly generates specific fragments, which can be separated by electrophoresis on agarose gels. The set of fragments from a specific DNA that has been cleaved by action of a given restriction enzyme is constant for that DNA. By use of various restriction enzymes ‘Howard Hughes Medical Institute, and R. J. Kleberg, Jr., Center for Human Genetics, at Baylor College of Medicine, Houston, TX 77030. 2Department of Pathology, The Woman’s Hospital of Texas, 7600 Fannin, Houston, TX 77054. Received October 23, 1984; accepted February 13, 1985. CLINICAL CHEMISTRY, of Terms Bacteriophage: Viruses that infect bacteria. Bacteriophage lambda is commonly used in studies with recombinant DNA. Chromosomal translocation: A rearrangement of chromosomal material such that the chromosome breaks and a fragment of it is joined to a different chromosome. Cosmid: Plasmids into which the “cos” site of bacteriophage lambda has been inserted. A cosmid also allows plasmid molecules to be inserted into viral coat particles in vitro. DNA ligase: An enzyme capable of covalently joining two ends of DNA molecules. DNA probe: A fragment of single-stranded DNA that can be labeled with a radioisotope or biotin and hybridized to an unknown DNA sample to determine whether the probe and the unknown DNA have sequences of nucleotides in common. Enhancer: DNA elements that can increase the efficiency of promoter regions. Gene: A segment of DNA that codes for a single polypeptide. Hybridization: The annealing of two strands of single-stranded DNA (or RNA) whose sequences of bases are complimentary sources reactions. Exposure to information, but DNA radical shifts in salt concentration, and high temperature, and it can be physically ensconced in a nitrocellulose matrix without losing its ability to hybridize by base pairing. This allows a cloned and radioactive DNA probe to recognize precisely its complementary base sequence by hybridization in a mixture of DNA or RNA isolated from cellular sources. This principle is the underpinning of DNA:DNA (homoduplex) or DNA:RNA (heteroduplex) hybridization reactions. 804 Diagnosis that recognize different internal sequences, specific patterns can be obtained for a particular DNA More than 140 restriction endonucleases are currently available. Use of a battery of these endonucleases, together with establishment of fragment overlaps, permits development of gene maps. fragment sample. I I I AAT III I I I GLAATT CIT I I II TC -3- I C AAIG I I I’ I CTTAA I Fig. 1. Restriction in “sticky ends” endonuclease cleavage I I I I of DNA by Eco RI, Often it is desirable to clone portions of DNA directly from the genome and study long stretches (20 kb) of DNA. A third vector system, “cosmids,” makes this possible. Cosmids are produced from “cos” sites taken from lambda phage DNA attached to portions of plasmid material-hence the name cosmid. These systems can accommodate genomic DNA or cDNA fragments of 20-45 kb. The efficiency of cosmid cloning is high, and allows examination of longer sequences of DNA. Organically synthesized DNA probes can also be made if protein or gene sequence data are available for the gene of interest. resulting Southern of DNA Probes technical The molecular biologist has two sources for isolating cloned DNA probes: the native DNA in the nucleus of a cell (genomic DNA), and complementary DNA (cDNA), made by enzymically (with reverse transcriptase) copying messenger RNA (mRNA). DNA probes can be generated by cloning procedures (2) by use of restriction endonuclease fragments inserted into bacterial plasmids. Plasmids are circular DNA molecules that replicate independently, encode antibiotic-resistance genes, and thus confer antibiotic resistance in the host bacterium. Plasmids carrying fragments of foreign DNA can be constructed by recombinant DNA methods by using a restriction endonuclease and DNA ligase. A restriction enzyme can cleave the plasmid and the foreign DNA, yielding identical complementary singlestranded junctions (“sticky ends”), which the DNA ligase enzymically links covalently. Because a single molecule of the recombinant plasmid can be replicated in 50 to 200 copies in proliferating Escherichia coli, large quantities of the cloned DNA can be obtained. Cloned DNA inserts are recovered in abundance by being excised from the recombinant plasmid by the same restriction enzyme used in making the construction (Figure 2). Plasmids efficiently replicate with DNA inserts that are 1000 to 6000 base pairs (one to six kilobases, kb) long. Larger fragments are not reliably replicated. An alternative approach, which makes use of bacteriophage lambda, allows larger fragments-up to about 20 kb-to be efficiently cloned. The fragments of DNA (or cDNA) to be cloned are inserted into modified phage, which are then used to infect the bacteria. Bacteriophage replicates in the bacteria and is subsequently harvested by lysing the cells. and isolated molecule Foreign 1975, E. M. Southern (3) developed a widely adopted procedure for transferring electrophoretically separated DNA species to nitrocellulose (Figure 3). Once immobilized in nitrocellulose, DNA can be analyzed for specific nucleotide sequences by use of labeled cloned DNA probes. The advantage of Southern’s approach is that the resolving power of gel separation is efficiently combined with the specificity of DNA probes. This procedure (3,4) involves use of standard electrophoretic agarose gel techniques. Treatment of the doublestanded DNA with dilute sodium hydroxide effects strand separation. Nitrocellulose or charged nylon ifiters are placed on the gel for strand transfer by buffered salt solution. Within hours, DNA fragments are transferred from the gel to the mtrocellulose ifiter. Very low-Mr species (fewer than 300 base pairs) are somewhat under-represented, and very large fragments (>15 000 base pairs) often take longer to transfer (3,5). As transfer occurs, the DNA molecules bind to the mtrocellulose. Hybridization with the radioactive probe is done after the ifiter is washed free of salt and then dried under reduced pressure. Several recent improvements in hybridization conditions have increased the sensitivity of detection and decreased the background. In Creation Transfer DNA is cloned with high Dot (Slot) Blotting A second technique used to probe DNA or RNA samples is “dot-blotting” (6, 7) or “slot-blotting.” This technique is used to detect the presence, absence, or amount of a genetic element. The technique requires neither size fractionation Cleavage Gene + Cleavage Locus vvl 4Kb cules. Ri Enzyme efficiency in large quantity, because a single recombinant suffices to form thousands of bacteriophage mole- Eco Sites for Restriction Iv 1Kb 7.2Kb $ Sites Cut with v 6Kb restriction v 3.5Kb enzyme 6Kb Cloned DNA insert or / cDNA 0 1Kb $ Gel Hybridize to P32 DNAwith cut Eco Ri 7.6Kb - 6Kb Labelled Cloned 4Kb 3.5Kb ReplIcation in Bacteria PIaenld DNA Fig. 2. Plasmid cloning of foreign DNA or cDNA by annealing of the ends” produced by Eco RI “sticky DNA 1Kb Probe DNA+Fluorescent Stain Radioautogram Fig. 3. Southem gel method from a DNA mixture (3) for identification of a gene fragment CLINICAL CHEMISTRY, Vol. 31, No. 6, 1985 805 nor cleavage with enzyme. Samples of DNA or RNA are extracted and dotted onto nitrocellulose ifiters or, to save space, applied as slots 2 mm wide x 5 mm long. Hybridization is carried out as for Southern blotting, and probes are detected by autoradiography. With radiolabeled probes of high specific activity, the resulting sensitivity (i.e., the smallest detectable amount) is about 0.2 to 0.5 pg (6; G. Buffone, personal communication). This is an especially valuable technique for use with microgram quantities of samples (6) and for detection of viral sequences in a sample (8-10). In Situ Hybridization A third technique based upon the use of DNA probes is in situ hybridization, used to hybridize probes to chromosomes (11,12) or tissue sections (13,14). Its usefulness results from its ability to localize relevant DNA sequences in larger structures, thus linking biochemistry with cytogenetics or histochemistry. Genes can be localized to specific chromosomes by radioactive probes and the results made visible by autoradiography of the chromosomes. Similarly, important information about gene expression can be obtained in relation to the development of specific tissues by identifying the presence of mRNA in histological sections by autoradiography or fluorescence microscopy. Viral genomes can be specifically localized within tissues with this technique (14). Tissues are fixed in Carnoy’s B solution (ethanollchlorofonn/ acetic acid, 60/30/10 by vol), embedded in paraffin, and sectioned as usual. The hybridization is performed directly on the cut sections. DNA probes used for in situ cytohybridization (in tissues) can be either radiolabeled for autoradiography or biotinylated (13, 14) and coupled to fluorescent or immunoenzymic detection systems (Figure 4). The latter technique has potential for anatomical and clinical pathology laboratories, because no radioisotopes are required to detect the viral genome in tissue sections or fluid specimens such as urine. Of the various potential immunoenzymic systems, peroxidase (EC 1.11.1.7)-anti-peroxidase and alkaline phosphatase (EC 3.1.3.1)-anti-alkaline phosphatase (15) are the most widely used. Biotin, covalently linked to triphosphates of thymidine, uridine, or cytidine (16), works most effectively as a “recorder” molecule if it is separated by about 1 nm from the nucleotide by an 11- to 16-carbon spacer-arm (13). Biotinylated nucleotides are then incorporated into the DNA probe by using the enzyme DNAdirected DNA polymerase (EC 2.7.7.7). This same procedure can be used to incorporate radiolabeled nucleotides, generating highly radioactive probes, with specific activities exceeding i0 counts/mm per microgram. AIkaIIns Phosphata.. Moses Anti-alkallns Shssp AntI- Mouss AntI-blotln Blotlnylat#{149}d I AC G Phosphatass Moses Probs I II I Fig. 4. Immunoenzymic detection complementary biotinylated probe, tion of a visually detected product 806 CLINICAL CHEMISTRY, Heritable Disease Recombinant DNA technology is currently being applied in clinical medicine to heritable, neoplastic, and infectious diseases. The diagnosis and understanding of inherited diseases have benefitted from the use of restriction endonucleases and DNA probes. DNA or RNA samples can be prepared from biopsies of chorionic villus or from amniocytes obtained by amniocentesis. Fibroblasts cultured from persons suspected of having an inherited disease can be used antenatally. Diagnosis by use of this material and DNA hybridization is limited only by the availability of cloned or synthetically derived probes. Recently developed probes include ones for Lesch-Nyhan trait (17), a1-antitrypsin (18), phenylketonuria (19), and deficiencies of argininosuccinate lyase (20), clotting factor IX (21), and insulin (22). Lesch-Nyhan disease results from a deficiency of hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8), an essential enzyme in purine metabolism. Such patients show mental retardation, extrapyrainidal choreoathetoid spasticity, and a compulsive tendency to self-mutilation. Hyperuricemia and increased urinary excretion of uric acid are constant findings. A recently developed probe for the HPRT gene (17) can be hybridized to fibroblast DNA (Figure 5, A and B) to screen for deletions of all or a portion of the gene responsible for the Lesch-Nyhan syndrome. This probe can also be used in cases of Lesch-Nyhan syndrome in which the only abnormality of the mutant gene is a point mutation (i.e., a single nucleotide substitution). The HPRT probe can also be used to study the mRNA of the mutant gene (Figure SC) (23). Sequencing procedures (24,25) are then used to determine the incorrect base substitution. In some genetic diseases the site of the mutation is constant. Knowledge of the normal sequence at the site of the sickle cell mutation allowed Chang and Kan (26) to use restriction endonuclease Mstll for diagnosis. The sickle cell mutation involves a single base change in a single codon, from GAG to GTG. Mstll recognizes the base sequence containing GAG, but not GTG. Treatment of DNA with MatH therefore provides fragments that are of different lengths for sickle cell homozygotes (SA), heterozygotes (SA), and normal homozygotes (AA): homozygotes of sickle cell disease (SS) show fragments containing 1350 base pairs, AA (normals) 1150, and sickle cell heterozygotes (SA) both sizes (26). Knowledge of the normal sequence at a mutation site also allows organically synthesized probes to be used for diagnosis. Nucleotides 19 bases long have been synthesized for the mutation site of the normal (A) and the sickle gene (5) and can discriminate between the normal and affected individuals(27). Synthesized probes are also useful for studying diseases where the amino acid substitution is known but gene structure data are unavailable. By this method Woo et al. (28) have been able to distinguish between the M and Z alleles in a1-antitrypsin deficiency. By this approach, heterozygotes can be accurately differentiated from nonnal individuals at the genetic level. Orkin and Markham (29) have successfully used synthetic probes to diagnose a thalassemia variant in which a single base change leads to abnormal processing of J3-globin mRNA. Gene probes, both synthesized and cloned, provide powerful tools for genetic disease diagnosis, but they require a specific knowledge of the gene or enzyme involved. Another ATGCA I Applications of a DNA sequence by use of a with alkaline phosphatase produc- Vol. 31, No. 6, 1985 approach, restriction fragment length polymorphisms (RFLPs), is applicable to illnesses where information about the gene, or even about the chromosome involved, is lacking ‘A C Exon 1+2 Exon 3 I 9.4Kb - Kb -6.6 -22 e#{248}n 4’ Lion Kb 4.4 Kb xs78,9- -1O Eson ‘2.3 Kb “-20 Kb brns 1,35Kb FIg. 5. Malysis of Lesch-Nyhan (A) Deletion of exons 6,7,8, muta ons by method of Southern and 9 (P.JK849); and Northern (2 exons 7,8, and 9 (RJK3487); and complete gene (RJK853). The DNA was digested with endonuclease Pst. (B) Partial by new BgI I fragments with exon-specific probes. (C) The dl5erent mRNA charactaristicsof three Leech-Nyhan mutants, gene duplication,exons 2 and 3, are detected shown by Northern analysis. All patients lacked HPRT enzymlc activity (Figure 6) (30). The sites recognized by restriction enzymes are interspersed throughout the human genome. Differences between individuals in the type, number, and position of these sites provide an abundant source of genetic markers for linkage studies. DNA fragments of variable size are therefore generated when DNAs from different individuals are cleaved with the same restriction enzyme and identified by the Southern method with a single probe. These variations in fragment size, RFLP, are inherited. In families where a genetic disease is expressed, it is possible, A. 13.0Kb Il) as V V V aA 7.6Kb B. AS Mother AS SS AA Father Child Child Fetus1 13.0 7.6 Fig. 6. Restriction fragment length polymorphism linked to the sickle cell gene (A) Diagram of restriction sites in DNA fiarwcing a p5 and p5 gene. The p-globln genes are found in 13 and 7 kb restriction fragments, respectively. (0) Southern analysis of DNA digested with the indicated endonuclease and probed with the globin gene (boxed area). The globin genotypes of the patients by the RFLP linkage with the A and S genes predicted and fetus are by using an appropriate probe, to link the occurrence of a particular RFLP pattern with the disease gene. By performing RFLP studies on large numbers of family members,’ affected and unaffected, the degree of probability that the’ abnormal RFLP pattern will occur when the disease gene is’ present can be statistically assessed. This probability dofines the degree of “linkage” between the disease gene and the occurrence of the abnormal fragment pattern. This knowledge can be used in subsequent RFLP studies to determine which family members are asymptomatic carriers of the gene and can, in some cases, be extended to antenatal diagnosis. Thus RFLP studies are useful for genetic counseling as well as disease diagnosis. This approach has been successfully exploited with several hemoglobinopathies, including the /3-thalassemias (3133). The f3-globin gene cluster, located on the short arm of chromosome 11 is represented by several different genetic abnormalities in the clinical group of p-thalassemiss, including abnormalities in RNA transcription (34), RNA processing (36), and RNA translation (37). Phillips et al. (38) used RFLPs to identifr polymorphisms associated with the y.globin genes and used these in the prenatal diagnosis of sickle cell anemia and p.thalassemia mutations. This has been a successful prenatal diagnostic technique in more than 100 families (39). Probes to known genes or probes to DNA sequences of no known function (“anonymous sequences”) can be used in RFLP linkage studies. RFLP techniques combined with in situ chromosomal hybridization can determine which chromosome carries the fragment closely linked to the disease gene. Recently, Gusella et al. (40) screened Venezuelan and American families with Huntington’s disease for RFLP linkages. Restriction digests of DNA from affected and normal family members were probed with anonymous sequences to uncover associations between the Huntington’s disease gene and various different probes. Although Gusella had originally estimated that as many as 100 anonymous sequence probes might have to be tested to find a reliable linkage (41), fortuitously, the twelfth probe (“G8”) provided CLINICAL CHEMISTRY, Vol. 31, No. 6, 1985 807 evidence of linkage; in situ hybridization then localized G8 to chromosome 4(40). The degree of linkage between G8 and the Huntington’s gene is quite high. Additional studies will doubtless lend to improved knowledge of linkage by isolating the DNA adjacent to the G8 clone and closer to the Huntington’s gene. Continued development should lead to a clinically useful method for detection of the Huntington’s gene (41) in families at risk. This example illustrates the power of RFLP linkage studies applied to genetic diseases about which the nature of the protein and gene defects is completely unknown. Given the high degree of RFLP reported for both phenylketonuria (43) and Lesch-Nyhan syndrome (43), RFLP analysis of affected families should improve carrier detection and prenatal diagnosis for both diseases. The gene for Duchenne’s muscular dystrophy has been mapped to band 21 of the short arm of the X chromosome (Xp2l), and probes are available that map to that region (44). Prenatal diagnosis for Duchenne’s is predictably going to be available as probes with tighter linkage to the Duchenne’s gene become available. Neoplastic Disease Recombinant DNA techniques have not yet made a formal impact upon the clinical diagnosis of neoplastic disease but this seems inevitable. Already these techniques have expanded the scope of understanding of the pathogenetic nature of the neoplastic process. The discovery and current understanding of oncogenes is a case in point. The concept of oncogenes derives from two independent lines of investigation. Studies of chemically induced carcinogenesis identified specific genetic elements capable of inducing neoplastic transformation in fibroblasts that took up DNA from transformed cells (45-47). Second, genes with transforming properties, found in certain retroviruses (46, 48), were termed oncogenes. Both lines of study make it clear that the genes are not in themselves a sufficient condition for transformation but require a given biologic context to operate-in keeping with the idea that cancer occurs as a multistep process. Oncogenes correspond to naturally occurring genes in the host genome, “proto-oncogenes,” which seem to be important in the control of embryonic development, differentiation, and cell growth. Some proto-oncogenes produce proteins similar to components of receptors for growth factors such as epidermal growth factor (49) and platelet-derived growth factor (50). More than 30 oncogenes have been identified (46). Neoplastic transformation may occur when the amounts of genetic transcripts from oncogenes are abnormally high (46). Gene amplification (the production of multiple copies of the same gene) and abnormal regulation of gene transcription are two ways this might happen. An oncogene inserted into host DNA in the region of a powerful “promoter” or “enhancer” could result in high proportions of transcription with correspondingly increased amounts of gene product. Similarly, proto-oncogenes may be juxtaposed to such a region secondary to chromosomal translocation (51). This is thought to be the mechanism by which, e.g., the myc oncogene is activated in Burkitt lymphoma. Alternatively, transformation may result if a point mutation in a protooncogene produces an amino acid substitution in a critical position in its gene (i.e., rasH) (46). Altered protein might disrupt critical processes in the cell, leading to features of neoplastic transformation: e.g., altered adhesion properties, abnormal growth, and shape. Circumstantial evidence supports these general mechanisms. For some time we have known that many neoplastic 808 CLINICAL CHEMISTRY, Vol. 31, No. 6, 1985 diseases are associated with chromosomal aberrations (52, 53), including Burkitt lymphoma, Philadelphia (Ph) chromosome-positive cases of chronic myelocytic leukemia, acute promyelocytic leukemia, retinoblastoma, and Wilma’s tumor. Recombinant techniques are extending an understanding of the chromosomal aberrations to the level of molecular genetics (51, 54). For other tumors, molecular genetic abnormalities have been identified where no previous cytogenetic lesion was known. Chromosomal translocation is relevant to induction of neoplasia in Burkitt lymphoma and most cases of chronic myelocytic leukemia. For Burkitt lymphoma, often associated with a translocation (55, 56) of the distal long arm of chromosome 8 to one of three other chromosomes (2, 14, or 22), the breakpoint of the translocated fragment is consistently at band 24 of the long arm of chromosome 8. DallaFavera et al. (57), studying somatic cell hybrids containing portions of chromosome 8 from Burkitt lymphoma cell lines, used a DNA probe for the oncogene c-myc that was homologous with the transforming gene of avian myelocytomatosis virus (v-myc). Their Southern blot analyses confirmed that c-myc is situated on chromosome 8. Taub et al. (58) and others (59) further refined the c-myc location to the band 24 translocation site. The majority of cases of Burkitt lymphoma involve a translocation from chromosome 8 to the immunoglobulin heavy chain loci on the long arm of chromosome 14 (58), but a significant minority are associated with translocations to the immunoglobulin kappa light chain gene on the short arm of chromosome 2 (51, 60). Some have hypothesized (51,58-60) that such translocations may bring about neoplastic transformation by juxtaposing the c-myc gene on chromosome 8 next to a promoter or enhancer region for immunoglobulin genes on chromosomes 14, 2, or 22. This would bring about an abnormal regulation of the oncogene and subsequent neoplastic transformation of the cell. The case for Ph chromosome-positive patients with chronic myelocytic leukemia is analogous. Patients with the Ph chromosome constitute about 92% of the adult patients with chronic myelocytic leukemia (62); they have a better prognosis than Ph chromosome-negative patients. The translocation involved is from chromosome 9 to, most commonly, chromosome 22. Such translocations were studied by DeKlein et al. (61), using in situ chromosome hybridization; they localized the oncogene c-abl to the breakpoint on the long arm of chromosome 9. The c-abl oncogene is homologous to the transforming gene of Abelson murine leukemia virus; thus, the hypothesis of abnormal regulation of an oncogene at a translocation breakpoint can be invoked here also. A 15/17 translocation has been identified in patients with acute promyelocytic leukemia (M3 subtype) of either the common hypergranular type or microgranular variant (51, 62-64). Evidence implicating specific oncogenes in this disease awaits further studies. Oncogenes appear to act dominantly when activated by translocations. However, recessive tumorigenic alleles may be operative in at least two childhood cancers. Childhood retinoblastoma (65, 66) and Wilma’s tumor (67) have both hereditary and sporadic forms. Hereditary transmission of retinoblastoma is autosomal dominant, but sporadic cases are associated with a cytogenetic deletion in the long arm of chromosome 13. The presumptive locus for the retinoblastoma gene is termed Rb-i (66). Wilma’s tumor is also sometimes associated with abnormalities of the short arm of chromosome 11. Several groups (65, 68-71) have now compared normal tissue DNA and tumor DNA by Southern blot analyses after digestions with various restriction endonucle- ases and the use of cloned DNA probes to genes in the vicinities of the known chromosomal deletions. The results tend to support the hypothesis that a recessive mutant gene is involved in some sporadic cases for both tumors. Expression of a recessive tumorigenic gene requires that it be present as the only allele in the tumor tissue. This could occur if the wild-type or normal allele were lost through mitotic non-disjunction or recombination (65, 69, 71). The mutant allele could then be duplicated locally to produce a homozygous state. In Wilma’s tumor, gene locus deletions have been demonstrated when there is no visible cytogenetic abnormality (68-70). Viral genomic probes are providing intriguing associations between other neoplasms and several types of viruses. Probes of Epstein-Barr virus DNA (72, 73) have added stronger evidence on the association of this virus and lymphomas. Hochberg et al. (72) demonstrated by gel separation-blotting procedures the presence of the viral genome in brain lymphoma tissue but not in adjacent uninvolved brain. Lancaster et al. (74), using similar methods, demonstrated a relationship between papilloma virus infection and dysplasias of the uterine cervix. Lass and associates (75) found the papilloma virus genome in conjunctival papillomas. Human T-cell leukemia-lymphoma virus type I (HTLV-I) is the first retrovirus to be convincingly associated with a human malignancy (76), although other retroviruses have an established association with several animal neoplasms. Evidence from recombinant methods and standard virologic techniques has implicated HTLV-I in the pathogenesis of a subtype of T-cell leukemia (76). This adult illness manifests splenomegaly, hypercalcemia, and skin disorders (77). Geographic concentrations occur in Japan, South America, the Caribbean, and some pai-ts of Africa. Dot blot procedures involving 2 zg of DNA can efficiently screen for one viral genome in fewer than 106 molecules (76). This technique is therefore useful both for diagnostic studies and the epidemiological surveys necessary to pinpoint high-density locales. Infectious Disease DNA probes are now available for many viral genomes, including hepatitis B, Epstein-Barr, herpes simplex types I and II, papova-viruses (BK, JC, and 5V40), papilloma viruses of several subtypes, adenoviruses, HTLV, and others. Recombinant techniques allow direct access to the viral genome. Not only can the presence of viral nucleic acid be assessed, but also information regarding the state of the genome can be obtained. By using appropriate restriction endonucleases and Southern blotting, one can determine whether viral DNA is present in a cell as an episome (viral DNA separate from host DNA) or is integrated into the host genome (78). if the viral DNA is integrated, partial viral genomic probes can be used to determine what portions of viral DNA have been inserted and which if any have been deleted. Such information is not possible with standard culture procedures and may elucidate the mechanisms of quiescent states in viral life cycles. Diagnostic virology makes use of available probes for rapid determination of the presence of pathogenic viruses, dot blot or slot blot hybridization being probably the most efficient type of procedure for this. Only microgram quantities of sample (host DNA) are required to detect picogram quantities of viral DNA (G. Buffone, personal communication). Southern blotting analysis must be applied after restriction enzyme digestion, to determine whether the viral genome has been integrated into the host DNA, but this requires larger amounts of DNA. The meaning of viral integration into the host genome relative to pathogenic states is not altogether clear at this point. Detection of viral nucleic acid in a sample is not synonymous with active production of virus particles within the cell. Thus DNA hybridization methods for viral detection, based on use of genomic digests, must be assessed carefully. Production of virions-detectable by immunochemical methods or electron microscopy-is therefore more reliable direct evidence of viral replication. An interesting application of hybridization technology relative to viral diseases is in situ cytohybridization (13,14). Even with radiolabeled probes this procedure is not as sensitive as dot blot analysis for indicating the presence of a viral genome, but it makes possible the direct tissue localization of virus material. Weller et al. (79) used this technique to demonstrate hepatitis B virus in hepatocytes, and Grinnell et al. (78) could localize the JC subtype of papovavirus to specific sites in patients dying from progressive multifocal leukoencephalopathy. This capability, like immunocytochemistry, allows a biochemical approach to tissue sections-of critical importance when biopsy material is small and cannot be divided for culture, biochemical, and morphologic studies. DNA in situ cytohybridization can even be performed with fixed and paraffin-embedded material, and combined with immunoenzymic detection systems. In cases of suspected herpes encephalitis or progressive multifocal leukoencephalopathy, this approach allows rapid specific diagnosis with minimum material and technical time. HTLV-ffl has been suggested as the etiologic agent in cases of acquired immunodeficiency syndrome (AIDS) (8084). Antibodies against envelope and core antigens of HTLV-ffl have been shown to have value in the diagnosis of this disease (82,83,85). Commercial enzyme-linked immunoabsorbant and radioimmunoassay systems for detecting HTLV-IU are now under development (86). Recombinant DNA techniques may thus offer future diagnostic utility (87). This work was supported by Medical Service Program Grant Corp. Dev. 01-3 from the March of Dimes Birth Defects Foundation, Metropolitan Houston Chapter, to C.T.C., and by the Howard Hughes Medical Institute. References 1. Nathans D, Smith HO. Restriction endonucleases in the analysis and restructuring of DNA molecules. Annu Rev Biochem 44, 273293 (1975). 2. Maniatis T, Frit.sch EF, Sambrook J. Molecular Cloning: A Laboratoiy Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. 3. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503-517 (1975). 4. Southern Methods 5. Bittner EM. Enzymol Gel electrophoresis of restriction (1980). P, Morris CF. Electrophoretic fragments. 68, 152-176 M, Kupferer and nucleic acid from slab-gels onto paper or nitrocellulose sheets. Anal Biochem protein transfer of diazobenzyloxymethyl 102, 459-471 (1980). 6. Kafatos FC, Jones CW, Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nuci Acids Res 7, 1541-1552 (1979). 7. Brandama J, Miller G. Nucleic acid spot hybridization: Rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc Nail Acad Sci USA 77, 6851-6855 (1980). & Martin DC, Katzenstein DA, Yu GSM, Jordan MC. Cytomegalovirus viremia detected by molecular hybridization and electron microscopy. Ann Intern Med 100, 222-225 (1984). 9. Kam W, Rail LB, Smuckler BA, et al. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Natl Aced Sci USA 79, 7522-7526 (1982). CLINICAL CHEMISTRY, Vol. 31, No. 6, 1985 809 10. Geever RF, Wilson LB, Nallaseth FS, et al. Direct identification of sickle cell anemia by blot hybridization. Proc NatlAcad Sci USA 78, 5081-5085 (1981). 11. Harper ME, Ulirich A, Saunders GF. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83, 431-439 (1981). 12. Trent JM, Olson S, Lawn RM. Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of chromoeomal in situ hybridization. Proc Nat! Aced Sci USA 79, 7809-7813 (1982). 13. Brigati DJ, Myerson D, Leary JJ, et al. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. ViroLogy 126, 32-SO (1983). 14. Leary JJ, Brigati N, Ward DC. Rapid and sensitive colorimet. nc method for visualizing biotin-labelled DNA probe hybridized to DNA or RNA immobilized on mtrocelluloee: Bio-blota. Proc Nat! Aced Sci USA 80, 4045-4049(1983). 15. Cordell JL, Brunangelo F, Ether WN, et al. Ixnmunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32,219-229(1984). 16. Langer PR. Waldrop AA, Ward DC. Enzymatic synthesis of biotin-labelled polynucleotides: Novel nucleic acid affinity probes. Proc Nat! Aced Sci USA 78, 6633-6637 (1981). 17. Brennand J, Chinault AC, Konecki DS, et al. Cloned CDNA sequences of the HPRT gene from a mouse neuroblastoma cell line found to have amplified genomic sequences. Proc Nail Aced Sci USA 79, 1950-1954 (1982). 18. Kurachi K, Chandra T, Degen SJF, et al. Cloning and sequence of eDNA coding for a1-antitrypein. Proc Nail Aced Sci USA 78, 6826-6830 (1981). 19. Robeon KJH, Chandra T, MacGillivray RTA, Woo SLC. Polyaome immunoprecipitation of phenylalanine hydroxylaae mENA from rat liver and cloning of its eDNA. Proc Nat! Aced Sci USA 79, 4701-4705 (1982). 20. Su T-8, Bock H-GO, O’Brien WE, Beaudet AL. Cloning of cDNA for argininosuccinate synthetase mENA and study of enzyme overproduction in a human cell line. JBiol Chem 256,1132611331 (1981). 21. Wigmton DA, Adrian human adenosine 7485 (1983). GS, Friedman dearninase. Proc Nat! 22. Bell GI, Pictet RI, Rutter WJ. Sequence (London) 284, 26-32 (1980). gene. Nature RL, et al. Cloning of Sci USA 80, 7481- Aced of the human insulin DS, Brennand J, Fuacoe JC, et al. Hypoxanthineguanine phosphoribosyltransferase genes of mouse and Chinese hamster Construction and sequence analysis of cDNA recombinants. Nuci Acids Res 10, 6763-6775 (1982). 23. Konecki 24. Maxam 25. Sanger termination (1977). 26. Chang AM, Gilbert W. Methods Enzymol 65,499-560(1980). F’S, Nicklen Coulson AR. DNA sequencing with chaininhibitors. Proc Nat! Aced Sci USA 74, 5463-5467 JC, Kan YW. A sensitive new prenatal test for sickle N Engi J Med 307, 30-32 (1982). 27. Conner BJ, Reyes AA, Mona C, et al. Detection of sickle cell (a)-globin allele by hybridization with synthetic oligonucleotides. Proc Nat! Aced Sci USA 80, 278-282 (1983). 28. Woo SLC, Kidd VJ, Pam ZK, et al. In Banbury Report 14: Recombinant DNA Applications to Human Disease, Cl’ Caskey, RL White, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp 105-112. 29. Orkin S, Markham AF. Direct detection of the common fithalassemia gene by synthetic DNA probe: An alternative approach to prenatal diagnosis. J Clin Invest 71,775-779(1983). 30. Botatein D, White RL, Skolnick M, Davis RS. Construction of a cell anemia. genetic linkage polymorphism. 31. Weatherall map in man using restriction fragment length Am J Hum Genet 32, 314-331 (1980). N, Clegg JB. Thalassemia revisited. Cell 29, 7-9 (1982). 32. Orkin SH, Kazazian HH, Antonorakia SE, et al. Linkage of the fl-thalasaemia mutations and p-globin gene polymorphisms in human p-globin gene cluster. Proc Nat! Aced Sci USA 79,627-631 (1982). 810 CLINICAL CHEMISTRY, Vol. 31, No.6, 1985 33. Little PFR,, Annison G, DarlingS, et al. Model for antenatal diagnosis of p-tha]asaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms. Nature (London) 285, 144-151 (1980). A, Posakony JW, Maniatis T, et al. The structure and evolution of the human -globin gene family. Cell 21, 653 (1980). 35. Spnitz RA, Forget 8G. The thalassemias: Molecular mechanisms of human genetic disease. Am J Hum Genet 35, 333-361 (1983). 36. Orkin SH, Kazazian HH, Antonorakis SE, et al. Abnormal RNA processing due to the exon mutation epsilon)-globin gene. Nature (London) 300, 768-769 (1982). 37. Orkin SH, GOf SC. Nonsense and framesbift mutations in fl(o)thalassemia detected by cloned fl-globin gene polymorphisms with DNA polymorphisms in human -globin genes. J Biol Chem 256, 9782-9784 (1981). 38. Phillips JA. Danny SB, Kazazian HH, et al. Prenatal diagnosis of sickle cell anemia by restriction endonuclease analysis: Hindifi polymorphism in gamma-globin genes extend test applicability. Proc Nail Aced Sci USA 77, 2853-2856 (1980). 39. Boehm CD, Antonorakis SE, Phillips HA, et al. Prenatal diagnosis using DNA polymorphisms: Report on 95 pregnancies at risk for sickle cell disease or -thalassemia. N Engl J Med 308, 1054-1058 (1983). 40. Gusella JF, Wexler NS, Conneally PM, et al. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature (London) 306, 234-238 (1983). 41. Cantor CR. Charting the path to the Huntington’s gene. Nature (London) 308, 404-404 (1984). 42. Woo SLC, Lidsky AS, Guttler F, et al. Cloned human phenylal34. Efstradiatis anine hydroxylase gene permits detection of classical phenylketonuria. 155 (1983). prenatal diagnosis and carrier Nature (London) 306, 151.-. WE, Nyhan WL, Caskey CT. A three 43. Nuasbaum RL, Crowder allele restriction fragment polymorphism at the hypoxanthine phoephonibosyltransferase locus in man. Proc Nat! Aced Sci USA 80, 4035-4039 (1983). 44. Murray JM, Davies KB, Harper PS, et al. linkage relationship of a cloned DNA sequence on the short arm of the X chromosome to Duchenne muscular dystrophy. Nature (London) 300,69-71(1982). 45. Weinberg duced tumors. BA. Oncogenes Ado Cancer 46. Land H, Parada multistep carcinogenesis. LF, of spontaneous and Res 36, 149-163 (1982). chemically Weinberg A. Cellular oncogenes Science 222, 771-778 (1983). inand 47. Shih C, Shilp B, Goldfarb M, et al. Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. Proc Nat! Aced Sci USA 76, 5714-5718 (1979). 48. Weiss B, Teich N, Varmus H, Coffin J (Eds.). RNA Tumor Viruses, Cold Spring Harbor Laboratory Monograph Series, Cold Spring Harbor, NY, 1982. 49. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor receptor to v-erb-B oncogene protein sequences. Nature (London) 307, 521-527 (1984). 50. Waterfield MD, Scrace GT, Whittle N, et al. Platelet-derived growth factor is structurally related to the putative transforming protein p28(sis) of simian sarcoma virus. Nature (London) 304,3538 (1983). P, Battey J, Lenoir G, et al. Translocations among antibody genes in human cancer. Science 222, 765-771 (1983). 52. The Second International Workshop on Chromosomes in Leukemia. Cancer Genet Cytogenet 2,89-113 (1980). 53. Sandberg AA. The Chromosomes in Human Cancer and Leukemia, Elsevier/North Holland, New York, NY, 1980. 54. Yunis JJ. The chromoaomal basis of human neoplasia. Science 221, 227-236 (1983). 55. Manolova G, Manolova Y. Marker band in one chromosome from Burkitt lymphoma. Nature (London) 237, 33-34 (1974). 56. Berheim A, Berger B, Lenoir G. Cytogenetic studies on African Burkitta lymphoma cell lines: t(8;14), t(2;8) and t(8;22) translocations. Cancer Genet Cytogenet 3, 307-315 (1981). 51. Loden 57. Dalla-Favera B, Bnegni M, Erikson J, et al. Human c-myc one gene is located on the region of chromosome 8 that is translocated in lymphoma cells. Proc Nail Aced Sci USA 79, 7824-7827 (1982). 58. Taub T, Kirach I, Morton C, et al. Translocation of the c-myc gene into the iminunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc Nat! Aced Sci USA 79, 7837-7841 (1982). 59. Hollis GF, Mitchell KF, Battey J, et al. A variant translocation places the lambda immunoglobin genes 3’ to the c-myc oncogene in Burkitt lymphoma. Nature (London) 307, 752-755 (1984). 60. Bartram CR, DeKlein A, Hagemeiher A, et al. Translocation of c-abl oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukemia. Nature (London) 306, 277-280 (1983). 61. DeKlein A, Guerto Van Kessel A, Groaveld G, et al. A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature (London) 300, 765-767 (1982). Burkitt 62. Rowley JD, Golomb HM, Dougherty C. 15/17 translocation, a chromosomal change in acute promyelocytic leukemia. Lancet I, 549-550 (1977). 63. Van Den Berghe H, Louwagie A, Broeckaert-Van Orshoven A, et al. Chromosomal abnormalities in acute promyelocytic leukemia. Cancer 43, 558-562 (1979). consistent 64. Golonb HM, Rowley JD, Vardiman JW, et al. “Microgranular” acute promyelocutic leukemia: A distinct clinical, ultrastructural and cytogenetic entity. Blood 55, 253-259 (1980). 65. Cavenee WK, Dryja TP, Phiffips RA, et al. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature (London) 305, 779-784 (1983). 66. Murphree AL, Benedict WF. Retinoblastoma: Clues to human oncogenesis. Science 223, 1028-1033 (1984). 67. Knudson AG, Strong LC. Mutation and cancer A model for Wilma’s tumor of the kidney. JNat! Cancerlnst 48,313-324(1972). 73. Andiman W, Gradoville L, Heston L, et al. Use of cloned probes to detect Epstein-Barr viral DNA in tissues of patients with neoplastic and lymphoproliferative diseases. J Infect Die 148, 967977 (1983). 74. Lancaster WD, Kurman RI, Sanz LE, et al. Human papillomavirus: Detection of viral DNA sequences and evidence for molecular heterogeneity in metaplasias and dysplasias of the uterine cervix. Inte,virology 20, 202-212 (1983). 75. Lass JH, Grove AS, Papale JJ, et al. Detection of human papillomavirus DNA sequences in cotjunctival papilloma. Am J Ophtho.lmol 96, 670-674 (1983). 76. Manzari V, Agliano AM, Gallo RC, Wong-Staal F. A rapid and sensitive assay for proviral sequences of a human retrovirus (HTLV) in leukemia cells. Leuk Res 7,681-686(1983). 77. Gallo BC, Wong-Staal F. Retroviruaea as etiologic agents of some animal and human leukemias and lymphoma. Blood 60,545557 (1982). 78. Grinnell BW, Padgett BL, Walker DL. Distinction of nonintegrated DNA from JC papovavirus in organs of patients with progressive multifocal leukoencephalopathy. JlnfectDi8 149,669675 (1983). 79. Weller WD, Fowler MJF, Morardino J, Thomas HC. The detection of HBV-DNA in serum by molecular hybridization. J Med Vim! 9, 273-280 (1982). 80. Popovic M, Sarngadharan MG, Reed E, Gab RC. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-ffl) from patients with AIDS and pre-is. Science 224, 497500 (1984). 81. Gallo BC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-ffl) from patients with AIDS and at risk for #{194}me. Science 224, 500-503 (1984). 70. Reeve AE, Houxiaux PJ, Gardner RJM, et al. Loss of a Harvey ras allele in sporadic Wilma’s tumor. Nature (London) 309, 174-176 (1984). 71. Fearson ER, Volgelestein B, Feinberg AP. Somatic deletion and duplication of genes on chromosome ii in Wilma’s tumour. Nature (London) 309, 176-178 (1984). 82. Schupbach J, Popovic M, Gilden B, et al. Serological analysis of a subgroup of human T-lymphotropic retrovirusea (HTLV-ffl) associated with AIns. Science 224, 503-506 (1984). 83. Sarngadharan MG, Popovic M, Brush L, et al. Antibodies reactive with human T-lymphotropic retroviruses (BTLV-ll1) in the serum of patients with me. Science 224, 506-509 (1984). 84. Schupback J, Sarngadharan MG, Gallo BC. Antigens on HTLV-infected cells recognized by leukemia and AIDS sera are related to HTLV viral glycoprotein. Science 224, 607-610 (1984). 85. Kalyanaraman VS, Cabradilla CD, Getchell JP, et al. Antibodies to the core proteins of lymphadenopathy associated virus (LAV) in patients with ms. Science 225, 321-324 (1984). 86. Budiansky S. AIDS: Test companies chosen. Nature (London) 310, 6 (1984). 72. Hochberg FH, Miller G, Schooley RT, et al. Central nervous system lymphoma related to Epstein-Barr virus. N Engl J Med 309, 746-748 (1983). 87. Gelmann EP, Popovic M, Blayney D, et al. Proviral retrovirus, human T-cell leukemia virus in two patients Science 220, 862-865 (1983). 68. Koufos A, Hansen MF, Lampkin BC, et al. Loss of alleles at loci on human chromosome 11 during genesis of Wilms’s tumor. Nature (London) 309, 170-172 (1984). 69. Orkin SH, Goldman DS, Sallan SE. Development of homozygosity for chromosome lip markers in Wilma’s tumor. Nature (London) 309, 172-174 (1984). CLINICAL CHEMISTRY, DNA of a with Vol. 31, No. 6, 1985 Awe. 811