The relationship between chromosome structure and function at a human telomeric region.

We have sequenced a contiguous 284,495-bp segment of DNA extending from the terminal (TTAGGG)n repeats of the short arm of chromosome 16, providing a full description of the transition from telomeric through subtelomeric DNA to sequences that are unique to the chromosome. To complement and extend analysis of the primary sequence, we have characterized mRNA transcripts, patterns of DNA methylation and DNase I sensitivity. Together with previous data these studies describe in detail the structural and functional organization of a human telomeric region.

Implications of FRA16A structure for the mechanism of chromosomal fragile site genesis.

Fragile sites are chemically induced nonstaining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. DNA sequences encompassing and including the rare, autosomal, folate-sensitive fragile site, FRA16A, were isolated by positional cloning. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)n repeat. This repeat was adjacent to a CpG island that was methylated in fragile site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting), which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis.

Physical map of the region containing the gene for Batten disease (CLN3).

CLN3 has been mapped genetically to 16p12, to the interval between D16S288 and D16S383, a sex-averaged genetic distance of 2.1 cM. Analysis of disease haplotypes for four microsatellite markers in this interval, D16S288, D16S299, D16S298, and SPN, has shown significant allelic association between one allele at each of these loci and CLN3. All four of the associated markers were used as nucleation sites in the isolation of genomic clones (YACs). A contig was assembled which contains 3 of the 4 associated markers and which confirmed the relative order of these markers. Marker D16S272 has been located on the physical map between D16S288 and D16S299. Restriction mapping has demonstrated the location of possible CpG islands. One gene, STP, has been localised on the YAC contig proximal to D16S298 and is therefore a candidate for CLN3. Other genes, including IL4R, SGLT2, and UQCRC2, have been excluded from this region.

Isolation of genes from the Batten candidate region using exon amplification. Batten Disease Consortium.

In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes.

Phenol sulfotransferases: candidate genes for Batten disease.

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis; JNCL) is an autosomal recessive neurodegenerative disorder, characterized by the cytosomal accumulation of autofluorescent proteolipopigments in neurons and other cell types. The Batten disease gene (CLN3) has not yet been identified, but has been mapped to a small region of human chromosome area 16p12.1-p11.2. We recently reported the fortuitous discovery that the cytosolic phenol sulfotransferase gene (STP) is located within this same interval of chromosome 16p. Since phenol sulfotransferase is expressed in neurons, can sulfate lipophilic phenolic compounds, and is mapped near CLN3, STP is considered as a candidate gene for Batten disease. YAC and cosmid cloning results have further substantiated the close proximity of STP and a highly related sulfotransferase (STM), encoding the catecholamine-preferring enzyme, to the CLN3 region of chromosome 16p. In this report, we summarize some of the recent progress in the identification of two phenol sulfotransferase genes (STP and STM) as positional candidate genes for Batten disease.

Overview of human repetitive DNA sequences.

This appendix contains brief descriptions of the most abundant classes of repetitive DNA in the human genome. The chromosomal distribution of these classes of repeats are shown for human chromosome 16.

An analysis of the distribution and dose response of chromosome aberrations in human lymphocytes after in vitro exposure to 137Cesium gamma radiation.

The chromosome aberration yield for human lymphocytes exposed in vitro to various doses of 137Cesium has been studied. Dicentric, total acentric, and excess acentric data were seen to follow a Possion distribution. Calculated total hits demonstrated over-dispersion which could possibly be accounted for by a greater occurrence of single-hit phenomena being repaired than two-hit exchange processes. The resulting distribution generally contained an under-representation of cells with odd numbers of hits and an over-representation of zero- and even-hit classes as compared with Poisson predicted values. The relationship between dicentric yield and dose received in rads was fitted to the linear-quadratic formula Y = alpha D + beta D2 for dicentrics, yielding values of (20.1 +/- 3.8) X 10(-4) (aberrations/cell)/rad and (1.89 +/- 0.75) X 10(-6) (aberrations/cell)/rad2 for alpha and beta respectively. A plot of percent 'normal' cells versus the dose in rads resembled cell survival curves and was fitted to the relation P(D) = 100 e-Y where Y = alpha D + beta D2 with alpha = (23 +/- 11) X 10(-4) rad-1 and beta = (8.3 +/- 2.5) X 10(-6) rad-2. A possible use of scoring 'normal' cells for purposes of biological dosimetry is presented.

Cloning, structural characterization, and chromosomal localization of the human orthologue of Saccharomyces cerevisiae MSH5 gene.

We have cloned and characterized the human orthologue of the Saccharomyces cerevisiae MutS homologue 5 (MSH5) cDNA, as well as the human gene that encodes the MSH5 cDNA, as a step toward understanding the molecular genetic mechanisms involved in the biological function of this novel human protein. The identified cDNA contains a 2505-bp open reading frame (ORF) that encodes an 834-amino-acid polypeptide with a predicted molecular mass of 92.9 kDa. The amino acid sequence encoded by this cDNA includes sequence motifs that are conserved in all known MutS homologues existing in bacteria to humans. The cDNA appears, on the basis of amino acid sequence analysis, to be a member of the MutS family and shares 30% sequence identity with that of S. cerevisiae MSH5, a yeast gene that plays a critical role in facilitating crossover during meiosis. Northern blot analysis demonstrated the presence of a 2.9-kb human MSH5 mRNA species in all human tissues tested, but the highest expression was in human testis, an organ containing cells that undergo constant DNA synthesis and meiosis. The expression pattern of human MSH5 resembled that of the previously identified human MutS homologues MSH2, MSH3, and MSH6-genes that are involved in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC). In an effort to expedite the search for potential disease association with this new human MutS homologue, we have also determined the chromosomal location and structure of the human MSH5 locus. Sequence and structural characterization demonstrated that MSH5 spans approximately 25 kb and contains 26 exons that range in length from 36 bp for exon 8 to 254 bp for exon 25. MSH5 has been mapped to human chromosome band 6p21.3 by fluorescence in situ hybridization. Knowledge of the sequence and gene structure of MSH5 will now enable studies of the possible roles MSH5 may play in meiosis and/or DNA replicative mismatch repair.

The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA concentrations of 200 micrograms/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800 micrograms/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 micrograms/ml (10(7) diploid human cells/ml agarose insert).

Identification and characterization of the mouse MutS homolog 5: Msh5.

We have identified and characterized the complete cDNA and gene for the mouse MutS homolog 5 (Msh5), as a step toward understanding the molecular genetic mechanisms involved in the biological function of this new MutS homologous protein in mammals. The Msh5 cDNA contains a 2502-bp open reading frame (ORF) that encodes an 833-amino acid protein with a predicted molecular weight of 92.6 kDa, which shares 89.8% amino acid sequence identity with the human hMSH5 protein. Northern blot analysis demonstrated the presence of a Msh5 mRNA approximately 2.9-kb in length, most abundantly expressed in mouse testis. Yeast two-hybrid analysis indicated that the mouse Msh5 protein positively interacted with the human hMSH4 protein-suggesting that Msh5 shares common functional properties with its human counterpart. Sequence and structural analyses show that the mouse gene Msh5 spans approximately 18 kb and contains 24 exons that range in length from 36 bp for exon 7 to 392 bp for exon 1. Structural comparison with the human hMSH5 gene revealed that all of the Msh5 internal exons, but not introns, are conserved in length with the human hMSH5. The Msh5 gene is located on mouse Chromosome (Chr) 17 in a location that is syntenic to the region of human Chr 6 harboring the hMSH5 gene. The identification and characterization of Msh5 will facilitate studies of the potential functional roles of this new member of the MutS family.

Identification of Alu-mediated deletions in the Fanconi anemia gene FAA.

Fanconi anemia (FA) is an autosomal recessive syndrome associated with hypersensitivity to DNA cross-linking agents and predisposition to neoplasia. Eight complementation groups (A-H) have been described, but the only FA genes cloned so far are FAC and FAA. We have recently identified 40 different germline mutations, including microdeletions, microinsertions, and point mutations in genomic DNA from 97 FA patients from the International Fanconi Anemia Registry (IFAR) by single-strand conformational polymorphism (SSCP) analysis. Interestingly, only one mutant allele was identified in many of these patients. Haplotype analysis with intragenic polymorphisms, as well as cDNA analysis of some patients suggested the presence of large deletions that would not be detected by SSCP analysis. In this study, we report the occurrence of Alu-mediated genomic deletions in FAA. Two different deletions of 1.2 kb and 1.9 kb were found. Both deletions include exons 16 and 17 and remove a 156-bp segment from the transcript causing a shorter in-frame message. Sequence analysis revealed that introns 15 and 17 are rich in partial and complete Alu repeats. There are at least four head-to-tail arranged Alu elements in intron 17 and one in intron 15, all oriented in the 3'-->5' direction. Sequence analysis of the deletions showed that the 5' breakpoints occurred at different sites in the same Alu element in intron 15, while the 3' breakpoints were located in different Alu repeats in intron 17. Numerous Alu repeats are present in FAA, suggesting that Alu-mediated recombination might be an important mechanism for the generation of FAA mutations.

Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.

In situ hybridization mapping of human chromosome 16: evidence for a high frequency of repetitive DNA sequences.

Fluorescence in situ hybridization (FISH) provides a rapid approach to regional localization of overlapping clone sets (contigs) developed by various fingerprinting approaches. We have used 70 cosmid clones derived from 48 different contigs, part of the developing contig map of chromosome 16 (Stallings et al., 1990, 1992a), to cytogenetically map an estimated 8.6 million base pairs (Mbp) of chromosome 16 DNA (approximately 8-9% total coverage). Although the majority of cosmid contigs hybridized to single sites on chromosome 16, a significant fraction (23%) hybridized to multiple regions on chromosome 16; a subset of these also hybridized to other human chromosomes. In most instances, clones that mapped to multiple locations were found to contain low-abundance repetitive DNA sequences. The FISH data presented here, coupled with published mapping data from somatic cell hybrids (Callen et al., 1992), permits independent verification of the integrity of chromosome 16 cosmid contigs. The order of clones derived by FISH agrees closely with the cell hybrid mapping data and can be correlated with chromosome bands and specific chromosomal translocation breakpoints.

Genomic organization and the 5' flanking region of the gamma subunit of the human amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) complex is made up of at least three different subunits alpha, beta, and gamma, which are developmentally regulated, selectively expressed, and variously up-regulated by steroid hormones. To understand mechanisms involved in regulation of the gamma subunit, we have determined the structure of the human gammaENaC gene. By 5' rapid amplification of cDNA ends, primer extension analysis, and nuclease protection assay, we identified transcription start sites in human brain, kidney, and lung. A human genomic library was screened and overlapping cosmid clones that span approximately 50 kilobases and contain the hgammaENaC gene were identified. The 5'-untranslated region is 141 bases long, and the translation start codon is contained within the second exon. The human gene spans at least 35 kilobases. The 5' end of the gene including portions of 5' flanking genomic DNA and the first intron are G + C rich and contain several CpG dinucleotides, consistent with a CpG island. The 5' flanking region contains no CCAAT or TATA-like elements but does contain two GC boxes as well as several putative transcription factor binding sites including AP-2, Sp1, CRE, PEA-3, and NF-IL6. This is the first description of the structural organization and the 5' flanking region of a member of the epithelial sodium channel complex.

The Huntington disease locus is most likely within 325 kilobases of the chromosome 4p telomere.

The genetic defect responsible for Huntington disease was originally localized near the tip of the short arm of chromosome 4 by genetic linkage to the locus D4S10. Several markers closer to Huntington disease have since been isolated, but these all appear to be proximal to the defect. A physical map that extends from the most distal of these loci, D4S90, to the telomere of chromosome 4 was constructed. This map identifies at least two CpG islands as markers for Huntington disease candidate genes and places the most likely location of the Huntington disease defect remarkably close (within 325 kilobases) to the telomere.

Refined physical mapping of chromosome 16-specific low-abundance repetitive DNA sequences.

Repetitive DNA sequences have been implicated in the origin of several disease phenotypes, including fragile X syndrome, myotonic dystrophy, and spinal bulbar atrophy. In addition, a complex family of chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been mapped by fluorescence in situ hybridization to regions of chromosome 16 that undergo breakage/rearrangement in acute nonlymphocytic leukemia (ANLL) cells. It has been hypothesized that these repetitive sequences are causally related to the chromosome rearrangements found in ANLL. Here, we further refine the mapping of CH16LAR sequences with respect to the ANLL inversion breakpoints, using a panel of somatic cell hybrids containing 51 different chromosome 16 breakpoints. These studies indicate that CH16LAR sequences at 16p13 are in close proximity to the ANLL short-arm breakpoint region. However, the region containing the highest density of CH16LAR sequences on the long arm appears to be distal to the region where the ANLL long-arm breakpoint has been mapped. These studies further show that CH16LAR sequences map in close proximity to FRA16D and FRA16A.

A case for evolutionary genomics and the comprehensive examination of sequence biodiversity.

Comparative analysis is one of the most powerful methods available for understanding the diverse and complex systems found in biology, but it is often limited by a lack of comprehensive taxonomic sampling. Despite the recent development of powerful genome technologies capable of producing sequence data in large quantities (witness the recently completed first draft of the human genome), there has been relatively little change in how evolutionary studies are conducted. The application of genomic methods to evolutionary biology is a challenge, in part because gene segments from different organisms are manipulated separately, requiring individual purification, cloning, and sequencing. We suggest that a feasible approach to collecting genome-scale data sets for evolutionary biology (i.e., evolutionary genomics) may consist of combination of DNA samples prior to cloning and sequencing, followed by computational reconstruction of the original sequences. This approach will allow the full benefit of automated protocols developed by genome projects to be realized; taxon sampling levels can easily increase to thousands for targeted genomes and genomic regions. Sequence diversity at this level will dramatically improve the quality and accuracy of phylogenetic inference, as well as the accuracy and resolution of comparative evolutionary studies. In particular, it will be possible to make accurate estimates of normal evolution in the context of constant structural and functional constraints (i.e., site-specific substitution probabilities), along with accurate estimates of changes in evolutionary patterns, including pairwise coevolution between sites, adaptive bursts, and changes in selective constraints. These estimates can then be used to understand and predict the effects of protein structure and function on sequence evolution and to predict unknown details of protein structure, function, and functional divergence. In order to demonstrate the practicality of these ideas and the potential benefit for functional genomic analysis, we describe a pilot project we are conducting to simultaneously sequence large numbers of vertebrate mitochondrial genomes.

Genomic organization and DNA sequence of the human catecholamine-sulfating phenol sulfotransferase gene (STM).

The human monoamine neurotransmitter-preferring phenol sulfotransferase (M-PST) plays an essential role in the sulfation of catecholamines, such as dopamine. The cDNA encoding M-PST has been reported, and we have recently identified cosmid clones from human chromosome 16p11.2 for this gene, STM. Plasmid subclones derived from the STM cosmid clones were subjected to dideoxynucleotide chain termination sequencing to determine the genomic organization and DNA sequence of STM. The gene encoding full-length STM is approximately 6.4 kb and contains 8 exons and 7 introns.