Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

Positional cloning and characterization of the human aniridia and murine small eye genes

Aniridia (AN) is a congenital, panocular disorder of the eye characterized by the complete or partial absence of the iris. The disease can occur in both the sporadic and familial forms which, in the latter case, is inherited as an autosomal dominant trait with high penetrance. The objective of this study was to isolate and characterize the genes involved in AN and Sey, and thereby to gain a better understanding of the molecular basis of the two disorders.^ Using a positional cloning strategy, I have approached and cloned from the AN locus in human chromosomal band 11p13 a cDNA that is deleted in two patients with AN. The deletions in these patients overlap by about 70 kb and encompass the 3$\sp\prime$ end of the cDNA. This cDNA detects a 2.7 kb mRNA encoded by a transcription unit estimated to span approximately 50 kb of genomic DNA. The message is specifically expressed in all tissues affected in all forms of AN, namely within the presumptive iris, lens, neuroretina, the superficial layers of the cornea, the olfactory bulbs, and the cerebellum. Sequence analysis of the AN cDNA revealed a number of motifs characteristic of certain transcription factors. Chief among these are the presence of the paired domain, the homeodomain, and a carboxy-terminal domain rich in serine, threonine and proline residues. The overall structure shows high homology to the Drosophila segmentation gene paired and members of the murine Pax family of developmental control genes.^ Utilizing a conserved human genomic DNA sequence as probe, I was able to isolate an embryonic murine cDNA which is over 92% homologous in nucleotide sequence and virtually identical at the amino acid level to the human AN cDNA. The expression pattern of the murine gene is the same as that in man, supporting the conclusion that it probably corresponds to the Sey gene. Its specific expression in the neuroectodermal component of the eye, in glioblastomas, but not in the neural crest-derived PC12 pheochromocytoma cell line, suggests that a defect in neuroectodermal rather mesodermal development might be the common etiological factor underlying AN and Sey. ^

Identification of a novel tumor suppressor gene on human chromosome 3p14-p12 involved in renal cell carcinoma

Nonpapillary renal cell carcinoma (RCC) is an adult cancer of the kidney which occurs both in familial and sporadic forms. The familial form of RCC is associated with translocations involving chromosome 3 with a breakpoint at 3p14-p13. Studies focused on sporadic RCC have shown two commonly deleted regions at 3p14.3-p13 and 3p21.3. In addition, a more distal region mapping to 3p26-p25 has been linked to the Von Hippel Lindau (VHL) disease gene. A large proportion of VHL patients develop RCC. The short arm of human chromosome 3 can, therefore, be dissected into three distinct regions which could encode tumor suppressor genes for RCC. Loss or inactivation of one or more of these loci may be an important step in the genesis of RCC.^ I have used the technique of microcell-mediated chromosome transfer to introduce an intact, normal human chromosome 3 and defined fragments of 3p, dominantly marked with pSV2neo, into the highly malignant RCC cell line SN12C.19. The introduction of chromosome 3 and of a centric fragment of 3p, encompassing 3p14-q11, into SN12C.19 resulted in dramatic suppression of tumor growth in nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data define the region 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC, to contain a novel tumor suppressor locus involved in RCC. We have designated this locus nonpapillary renal cell carcinoma-1 (NRC-1). Furthermore, we have functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo. ^

Origins and evolution of VNTR loci: The apolipoprotein B 3$\sp\prime$ VNTR

I studied the apolipoprotein (apo) B 3$\sp\prime$ variable number tandem repeat (VNTR) and did computer simulations of the stepwise mutation model to address four questions: (1) How did the apo B VNTR originate? (2) What is the mutational mechanism of repeat number change at the apo B VNTR? (3) To what extent are population and molecular level events responsible for the determination of the contemporary apo B allele frequency distribution? (4) Can VNTR allele frequency distributions be explained by a simple and conservative mutation-drift model? I used three general approaches to address these questions: (1) I characterized the apo B VNTR region in non-human primate species; (2) I constructed haplotypes of polymorphic markers flanking the apo B VNTR in a sample of individuals from Lorrain, France and studied the associations between the flanking-marker haplotypes and apo B VNTR size; (3) I did computer simulations of the one-step stepwise mutation model and compared the results to real data in terms of four allele frequency distribution characteristics.^ The results of this work have allowed me to conclude that the apo B VNTR originated after an initial duplication of a sequence which is still present as a single copy sequence in New World monkey species. I conclude that this locus did not originate by the transposition of an array of repeats from somewhere else in the genome. It is unlikely that recombination is the primary mutational mechanism. Furthermore, the clustered nature of these associations implicates a stepwise mutational mechanism. From the high frequencies of certain haplotype-allele size combinations, it is evident that population level events have also been important in the determination of the apo B VNTR allele frequency distribution. Results from computer simulations of the one-step stepwise mutation model have allowed me to conclude that bimodal and multimodal allele frequency distributions are not unexpected at loci evolving via stepwise mutation mechanisms. Short tandem repeat loci fit the stepwise mutation model best, followed by microsatellite loci. I therefore conclude that there are differences in the mutational mechanisms of VNTR loci as classed by repeat unit size. (Abstract shortened by UMI.) ^

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^

Population genetics of VNTR loci and their applications in evolutionary studies

Variable number of tandem repeats (VNTR) are genetic loci at which short sequence motifs are found repeated different numbers of times among chromosomes. To explore the potential utility of VNTR loci in evolutionary studies, I have conducted a series of studies to address the following questions: (1) What are the population genetic properties of these loci? (2) What are the mutational mechanisms of repeat number change at these loci? (3) Can DNA profiles be used to measure the relatedness between a pair of individuals? (4) Can DNA fingerprint be used to measure the relatedness between populations in evolutionary studies? (5) Can microsatellite and short tandem repeat (STR) loci which mutate stepwisely be used in evolutionary analyses?^ A large number of VNTR loci typed in many populations were studied by means of statistical methods developed recently. The results of this work indicate that there is no significant departure from Hardy-Weinberg expectation (HWE) at VNTR loci in most of the human populations examined, and the departure from HWE in some VNTR loci are not solely caused by the presence of population sub-structure.^ A statistical procedure is developed to investigate the mutational mechanisms of VNTR loci by studying the allele frequency distributions of these loci. Comparisons of frequency distribution data on several hundreds VNTR loci with the predictions of two mutation models demonstrated that there are differences among VNTR loci grouped by repeat unit sizes.^ By extending the ITO method, I derived the distribution of the number of shared bands between individuals with any kinship relationship. A maximum likelihood estimation procedure is proposed to estimate the relatedness between individuals from the observed number of shared bands between them.^ It was believed that classical measures of genetic distance are not applicable to analysis of DNA fingerprints which reveal many minisatellite loci simultaneously in the genome, because the information regarding underlying alleles and loci is not available. I proposed a new measure of genetic distance based on band sharing between individuals that is applicable to DNA fingerprint data.^ To address the concern that microsatellite and STR loci may not be useful for evolutionary studies because of the convergent nature of their mutation mechanisms, by a theoretical study as well as by computer simulation, I conclude that the possible bias caused by the convergent mutations can be corrected, and a novel measure of genetic distance that makes the correction is suggested. In summary, I conclude that hypervariable VNTR loci are useful in evolutionary studies of closely related populations or species, especially in the study of human evolution and the history of geographic dispersal of Homo sapiens. (Abstract shortened by UMI.) ^

Insight into the strand exchange reaction promoted by the RecA protein of {\it Escherichia coli\/}

In vitro, RecA protein catalyses the exchange of single strands of DNA between different DNA molecules with sequence complementarity. In order to gain insight into this complex reaction and the roles of ATP binding and hydrolysis, two different approaches have been taken. The first is to use short single-stranded deoxyoligonucleotides as the ssDNA in strand exchange. These were used to determine the signal for hydrolysis and the structure of the RecA-DNA complex that hydrolyses ATP. I present a defined kinetic analysis of the nucleotide triphosphatase activity of RecA protein using short oligonucleotides as ssDNA cofactor. I compare the effects of both homopolymers and mixed base composition oligomers on the ATPase activity of RecA protein. I examine the steady state kinetic parameters of the ATPase reaction using these oligonucleotides as ssDNA cofactor, and show that although RecA can both bind to, and utilise, oligonucleotides 7 to 20 residues in length to support the repressor cleavage activity of RecA, these oligonucleotides are unable to efficiently stimulate the ATPase activity of RecA protein. I show that the K$\sb{\rm m}\sp{\rm ATP}$, the Hill coefficient for ATP binding, the extent of reaction, and k$\sb{\rm cat}$ are all a function of ssDNA chain length and that secondary structure may also play a role in determining the effects of a particular chain length on the ATPase activity of RecA protein.^ The second approach is to utilise one of the many mutants of RecA to gain insight into this complex reaction. The mutant selected was RecA1332. Surprisingly, in vitro, this mutant possesses a DNA-dependent ATPase activity. The K$\sb{\rm m}\sp{\rm ATP}$, Hill coefficient for ATP binding, and K$\sb{\rm m}\sp{\rm DNA}$ are similar to that of wild type. k$\sb{\rm cat}$ for the ATPase activity is reduced 3 to 12-fold, however. RecA1332 is unable to use deoxyoligonucleotides as DNA cofactors in the ATPase reaction, and demonstrates an increased sensitivity to inhibition by monovalent ions. It is able to perform strand exchange with ATP and ATP$\lbrack\gamma\rbrack$S but not with UTP, whereas the wild type protein is able to use all three nucleotide triphosphates. RecA1332 appears to be slowed in its ability to form intermediates and to convert these intermediates to products. (Abstract shortened by UMI.) ^

{\it In vivo\/} induction of DNA changes in cervicovaginal epithelium by perinatal estrogen exposure

Epidemiological studies have associated estrogens with human neoplasm such as the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17$\beta$-estradiol (17$\beta$-E$\sb2)\rbrack$ and synthetic (diethylstilbestrol (DES)) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17$\beta$-E$\sb2$ treatment increased the nuclear DNA content of mouse cervicovaginal epithelium that preceded histologically evident neoplasia. In order to determine whether this effect was specific to 17$\beta$-E$\sb2,$ associated with chromosomal changes, and relevant to the human, female BALB/c mice were treated neonatally with either 17$\alpha$-estradiol (17$\alpha$-E$\sb2)$ and 5$\beta$-dihydrotestosterone ($5\beta$-DHT), both inactive steroids in adult reproductive tissue, or 17$\beta$-E$\sb2.$ Ten-day-old mice received pellet implants of 17$\beta$-E$\sb2,$ 17$\alpha$-E$\sb2,$ $5\beta$-DHT, or cholesterol. Seventy-day-old cervicovaginal tracts were examined histologically and flow cytometrically. 17$\beta$-E$\sb2$-treated animals were evaluated by fluorescent in situ hybridization (FISH) using a probe specific for chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was evaluated by FISH in cervicovaginal material from 19 DES-exposed and 19 control patients.^ $17\beta$-E$\sb2, 17\alpha$-E$\sb2$, and $5\beta$-DHT-induced dramatic developmental and histological changes in the cervicovaginal tract, including hypospadia, hyperplasia, and persistent cornification. The changes induced by 17$\alpha$-E$\sb2$ were equivalent to 17$\beta$-E$\sb2.$ Neonatal 17$\alpha$-E$\sb2$-induced adenosquamous cervicovaginal tumors at 24 months. 17$\alpha$-E$\sb2$ and $5\beta$-DHT significantly increased the nuclear DNA content over control animals, but at significantly lower levels than 17$\beta$-E$\sb2.$ DNA ploidy changes were highest (80%) in animals treated neonatally and secondarily with 17$\beta$-E$\sb2.$ Secondary 17$\alpha$-E$\sb2$ and $5\beta$-DHT administration, unlike 17$\beta$-E$\sb2,$ didn't significantly increase DNA content. Chromosome 1 trisomy incidence was 66% in neonatal 17$\beta$-E$\sb2$-treated animals. Trisomy was evident in 4 DES-exposed patients: one patient with trisomy of chromosomes 1, 7, and 11; one patient with chromosome 7 trisomy; and two patients with chromosome 1 trisomy. These data demonstrated the biological effects of 17$\alpha$-E$\sb2$ and $5\beta$-DHT were age-dependent, 17$\alpha$-E$\sb2$ was equivalent to 17$\beta$-E$\sb2$ and tumorigenic when administered neonatally, and histological changes were not steroid specific. Chromosomal changes were associated with increased nuclear DNA content and chromosomal changes may be an early event in the development of tumors in human DES-exposed tissues. ^

Functional analysis of anionic phospholipids in cell metabolism in {\it Escherichia coli\/}

The major goal of this work was to understand the function of anionic phospholipid in E. coli cell metabolism. One important finding from this work is the requirement of anionic phospholipid for the DnaA protein-dependent initiation of DNA replication. An rnhA mutation, which bypasses the need for the DnaA protein through induction of constitutive stable DNA replication, suppressed the growth arrest phenotype of a $pgsA$ mutant in which the synthesis of anionic phospholipid was blocked. The maintenance of plasmids dependent on an $oriC$ site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein. In addition, structural and functional requirements of two major anionic phospholipids, phosphatidylglycerol and cardiolipin, were examined. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for the naturally occurring phosphatidylglycerol. The requirement for phosphatidylglycerol was concluded to be more than maintenance of the proper membrane surface charge. Examination of the role of cardiolipin revealed its ability to replace the zwitterionic phospholipid, phosphatidylethanolamine, in maintaining an optimal membrane lipid organization. This work also reported the DNA sequence of the cls gene, which encodes the CL synthase responsible for the synthesis of cardiolipin. ^

A study of reduced penetrance retinoblastoma

Retinoblastoma is a pediatric tumor which is associated with somatic and inherited mutations at the retinoblastoma susceptibility locus, RB1. Although most cases of retinoblastoma fit the previously described 'two hit' model of oncogenesis, the molecular mechanisms underlying rare instances of familial retinoblastoma with reduced penetrance are not well understood. To better understand this phenomenon, a study was undertaken to uncover the molecular cause of low penetrance retinoblastoma in a limited number of families. In one case, a unique cryptic splicing alteration was discovered in the RB1 gene and demonstrated to reduce the level of normal RB1 mRNA produced. Penetrance in the large family known to carry this mutation is less than 50%. Data about the mutation supports a theory that reduced penetrance retinoblastoma is caused by partially functional mutations in RB1. In another family, three independent causes of retinoblastoma or the related phenotype of retinoma were indicated by linkage analysis, a finding unique in retinoblastoma research. A novel polymorphism restricted to Asian populations was also described during the course of this study. ^

A study of the {\it in vivo\/} role of myogenin

Myogenin is a member of the MyoD family of skeletal muscle specific bHLH transcription factors. All of the members of this family have been shown to initiate the muscle differentiation cascade in a variety of nonmuscle cell lines. Many of the properties of the MyoD family have been studied in vitro, but their in vivo roles had not yet been examined. In this thesis, I study the in vivo role of myogenin by creating mice that carry a mutation at the myogenin locus.^ Mice lacking the myogenin protein are born alive, but immobile. Histological examination showed that these mice are severely deficient in skeletal muscle; they show a reduction in the number and density of myofibers. In addition to the reduction in fiber number, these mice express lower levels of a variety of muscle-specific markers. The undifferentiated cells in the muscle forming regions of these mice do express some muscle-specific markers, indicating that these cells are determined but undifferentiated myoblasts. Additional studies show that the major muscle defect arises late in embryogenesis, at a time coincident with secondary myogenesis. Moreover, studies regarding the nature of the remaining myofibers indicate that they are representative of a normal population of myofibers, merely reduced in numbers. In addition, I studied the effects of combining the myogenin mutation with mutations in two other members of the MyoD family, MyoD and myf5. Mice mutant in myogenin + MyoD and myogenin + myf5 show no increase in the severity of the myogenin single mutation, as indicated by histological or molecular examination. These results reveal the unique and essential role of myogenin in mammalian skeletal myogenesis. ^

Mitotic mapping of a novel tumor suppressor locus on human chromosome 3q important in osteosarcoma tumorigenesis

Tumor-specific loss of constitutional heterozygosity by deletion, mitotic recombination or nondisjunction is a common mechanism for tumor suppressor allele inactivation. When loss of heterozygosity is the result of mitotic recombination, or a segmental deletion event, only a portion of the chromosome is lost. This information can be used to map the location of new tumor suppressor genes. In osteosarcoma, the highest frequencies of loss of heterozygosity have been reported for chromosomes 3q, 13q, 17p. On chromosomes 13q and 17p, allelic losses are associated with loss of function at the retinoblastoma susceptibility locus (RB1) and the p53 locus, respectively. Chromosome 3q is also of particular interest because the high percent of loss of heterozygosity (62%-75%) suggests the presence of another tumor suppressor important for osteosarcoma tumorigenesis. To localize this putative tumor suppressor gene, we used polymorphic markers on chromosome 3q to find the smallest common region of allele loss. This putative tumor suppressor was localized to a 700 kb region on chromosome 3q26.2 between the polymorphic loci D3S1282 and D3S1246. ^

Characterization and subchromosomal location of a novel tumor suppressor gene on human chromosome 7

Alterations in oncogenes and tumor suppressor genes (TSGs) are considered to be critical steps in oncogenesis. Consistent deletions and loss of heterozygosity (LOH) of polymorphic markers in a determinate chromosomal fragment are known to be indicative of a closely mapping TSG. Deletion of the long arm of chromosome 7 (hchr 7) is a frequent trait in many kinds of human primary tumors. LOH was studied with an extensive set of markers on chromosome 7q in several types of human neoplasias (primary breast, prostate, colon, ovarian and head and neck carcinomas) to determine the location of a putative TSG. The extent of LOH varied depending the type of tumor studied but all the LOH curves we obtained had a peak at (C-A)$\sb{\rm n}$ microsatellite repeat D7S522 at 7q31.1 and showed a Gaussian distribution. The high incidence of LOH in all tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on the 7q31.1. To investigate whether the putative TSG is conserved in the syntenic mouse locus, we studied LOH of 30 markers along mouse chromosome 6 (mchr 6) in chemically induced squamous cell carcinomas (SCCs). Tumors were obtained from SENCAR and C57BL/6 x DBA/2 F1 females by a two-stage carcinogenesis protocol. The high incidence of LOH in the tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on mchr 6 A1. Since this segment is syntenic with the hchr 7q31, these data indicate that the putative TSG is conserved in both species. Functional evidence for the existence of a TSG in hchr 7 was obtained by microcell fusion transfer of a single hchr 7 into a murine SCC-derived cell line. Five out of seven hybrids had two to three-fold longer latency periods for in vivo tumorigenicity assays than parental cells. One of the unrepressed hybrids had a deletion in the introduced chromosome 7 involving q31.1-q31.3, confirming the LOH data. ^