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Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.

Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication. We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product. Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair. Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity. Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development. HEX1 gene equivalents were found in all vertebrates examined. The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.

A physical map of human chromosome 19p has been constructed by fluorescence in situ hybridization of cosmids to metaphase chromosomes and sperm pronuclear interphases. The map spans approximately 20 Mb and was generated with 141 multiple, partially overlapping estimates of genomic distances for 79 intervals separating 80 sequentially ordered cosmid reference points. The average distance separating pairs of cosmids was 250 kb, with a range from 50 to 700 kb; 75% of the intervals were estimated to be less than or equal to 300 kb and only 8 intervals were between 500 and 700 kb. Cosmids positive for 33 genes or gene families and 5 polymorphic markers were included among the mapped elements. The fluorescence in situ hybridization map will be useful for furthering the integration of the physical and genetic maps of 19p and for placing newly identified markers within a few hundred kb of their neighbors.

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequences as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

The intron-containing gene for human ribosomal protein RPL23A has been cloned, sequenced, and localized. The gene is approximately 4.0 kb in length and contains five exons and four introns. All splice sites exactly match the AG/GT consensus rule. The transcript is about 0.6 kb and is detected in all tissues examined. In adult tissues, the RPL23A transcript is dramatically more abundant in pancreas, skeletal muscle, and heart, while much less abundant in kidney, brain, placenta, lung, and liver. A full-length cDNA clone of 576 nt was identified, and the nucleotide sequence was found to match the exon sequence precisely. The open reading frame encodes a polypeptide of 156 amino acids, which is absolutely conserved with the rat RPL23A protein. In the 5' flanking region of the gene, a canonical TATA sequence and a defined CAAT box were found for the first time in a mammalian ribosomal protein gene. The intron-containing RPL23A gene was mapped to cytogenetic band 17q11 by fluorescence in situ hybridization.

We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19 cosmids on other chromosomes is also reported. Colony hybridization identified 142 cosmids that contain sequences representing genes or DNA markers that map to chromosome 19. FISH mapping of these cosmids sublocalizes a total of 70 genes and DNA markers on chromosome 19, revises the previously published map assignments of 2 genes, and narrows the location of over 20 markers.

The highly conserved Saccharomyces cerevisiae RAD51 protein functions in both mitotic and meiotic homologous recombination and in double-strand break repair. Screening of the public cDNA sequence database for RAD51-like genes led to the identification of a partial sequence from a breast tissue library present in the I.M.A.G.E. (Integrated Molecular Analysis of Genes and their Expression) collection. An extended 1764-bp cDNA clone encoding an open reading frame of 350 amino acids was isolated. This clone showed significant amino acid identity with other human RAD51 homologs. The new homolog, named RAD51B, was mapped to human chromosome 14q23-q24.2 using a panel of human-hamster somatic cell hybrids and fluorescence in situ hybridization. Northern blot analysis demonstrated that RAD51B mRNA is widely expressed and most abundant in tissues active in recombination. Functions associated with known RAD51 homologs suggest a role for RAD51B in meiotic recombination and/or recombinational repair. 30 refs., 2 figs.

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57B1/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f ]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.

We have developed a method that identifies human chromosomes in human × hamster somatic cell hybrids and simultaneously bands these same metaphases. Other methods generally require separate slides for banding and detection of human chromosome material, making the precise characterization of human material difficult. Our procedure involves denaturing metaphase chromosomes, followed by in situ hybridization of biotinylated whole human DNA. Fluoresceinated avidin is then bound to the biotinylated DNA, staining the human chromosomes yellow-green when excited with UV light. Chromosome banding is achieved by staining the slides with DAPI and actinomycin D. The fluorescein and DAPI excite maximally at 488 and 355 nm and emit at 520 and 450 nm, respectively. This permits identification of the human material at one excitation wavelength and visualization of the banding patterns at another wavelength. With this procedure, we have successfully identified both intact and broken human chromosomes, as well as human material involved in human × hamster translocations. The results indicate that this procedure is more accurate and considerably more rapid than previous methods and can be routinely employed for the cytogenetic analysis of human × rodent hybrids.

We measured the frequency of sister chromatid exchanges (SCEs) in human and mouse peripheral lymphocytes using doses of bromodeoxyuridine (BrdU) ranging from 30 ΠM to 100 µM (human) and from 10 nM to 10 µM (mouse). Heparinized peripheral blood was obtained from five healthy nonsmokers and from six C57B1/6 male mice. The blood was stimulated with PHA (human) or lipopolysaccharide (LPS, mouse) and grown for the first of two cell cycles in BrdU. Metaphase chromosomes were denatured and exposed to a monoclonal antibody reactive to single-stranded DNA containing BrdU. A second antibody was used to label the first antibody with fluorescein, and propidium iodide was used as a counterstain. Second-division metaphases were thus differentially stained red to indicate DNA content and yellow-green to indicate the presence of BrdU. The results indicate that the baseline SCE frequency in human and mouse peripheral lymphocytes is 3.6 and 2.4 SCEs per cell per generation, and that in the human these frequencies are invariant at the lowest BrdU levels. This suggests that SCEs are an integral part of DNA replication, even in the absence of agents known to induce SCEs. The distribution of SCEs per chromosome was analyzed and found to be Poisson-distributed in all 24 murine cultures and in 25 of 36 human cultures. The distribution of SCEs per chromosome may be due to either species-specific chromosome packaging or to karyotypic differences between the species.

A series of compounds isolated on the basis of their mutagenicity in the Ames/Salmonella reversion assay were previously identified in fried beef and chemically synthesized for further evaluation. In this study three of these compounds were tested for genotoxic effects in the UV5 line of Chinese hamster ovary (CHO) cells, which is deficient in nucleotide excision repair. Both 2-amino-3,4-dimethyl-imidazo]4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) gave very weak responses for cell killing, hprt mutation induction and sister chromatid exchange. These effects occurred at doses in the range of 100-800 micrograms/ml (approximately solubility limit), and dose-dependent increases were not observed. Induction of chromosomal aberrations did not occur with either compound. Nor did either of these compounds produce differential cytotoxicity in normal CHO cells versus UV5 cells, indicating that potentially repairable DNA damage was not responsible for the observed cell killing. In contrast to these results, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), which constitutes greater than 90% of the mass of bacterial mutagens in beef, was strongly positive for all endpoints at doses in the range 1-3 micrograms/ml. PhIP also gave marked differential cytotoxicity (ratio of 6) and cell survival curves that were strongly dependent on repair capacity. Because PhIP is 50- to 300-fold less mutagenic than MeIQ and MeIQx in Salmonella TA1538, these results point to major differences between the bacterial and mammalian assays in terms of the relative potency of these food-related compounds.

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.

We have investigated the genotoxic effects of three different brands and three types of coffee (freshly brewed regular, instant regular and freshly brewed decaffeinated) in two mammalian systems: the Chinese hamster ovary (CHO) AUXB1 cell line and human peripheral lymphocytes. Sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) were used as the endpoints. Coffee was prepared according to the manufacturer's suggestions, and after cooling, administered to cultured cells at dilutions ranging up to 11% that of full-strength coffee. Each brand and type of coffee induced significant levels of SCEs and ERCs in AUXB1 cells. SCEs, but not ERCs, were induced in human peripheral lymphocytes. Bisulfite, which complexes with carbonyls and reduces their genotoxicity, significantly diminished the number of SCEs and ERCs found after administration of coffee. Catalase and peroxidase, enzymes that destroy hydrogen peroxide activity, had no significant effect upon the SCE and ERC frequencies in AUXB1 cultures treated with freshly brewed regular coffee. These experiments indicate that different brands and types of coffee have sufficient genotoxic activity to increase SCEs and ERCs at levels only a fraction of those normally consumed. 1,2-Dicarbonyls alone and peroxides alone do not appear to be responsible for the majority of SCEs and ERCs that were observed to be induced by dilute coffee.