Genomic organization of the murine neuro-d4 gene

As the first step toward obtaining the null mutation or knock out of the neuro-d4 gene, we isolated phages containing fragments of the gene from a mouse genomic library. The nucleotide sequence of a region of the gene more than 10 kb in size was determined.     

Genomic organization and sequence variation of the human integrin subunit alpha8 gene (ITGA8).

The integrin alpha8 is highly expressed during kidney and lung development. alpha8-deficient mice display abnormal renal development suggesting that alpha8 plays a critical role in organogenesis. Therefore, it would be of considerable interest to understand the genomic structure, localization and sequence variation of the alpha8 gene. Using FISH and genomic database analysis, we show that alpha8 gene maps to chromosome 10p13 and consists of >200 kbp organized into 30 exons. Examination of 47 individuals from two different ethnic groups (European and African descent) identified 286 varying sites. The diversity of alpha8 is comparable to that of other regions within the human genome. Eight of the varying sites were located in the coding regions: six resulted in nonsynonymous substitutions of which two lead to non-conservative changes in protein. None of the sites showed significant deviation from Hardy-Weinberg equilibrium. We mapped the coding region single nucleotide polymorphisms (SNPs) onto a model of the predicted alpha8 structure and found all the SNPs were located in the "calf" of the extracellular domain. In the European population, the linkage disequilibrium statistic D' showed three blocks of relatively non-recombinant regions in the alpha8 gene while the African population showed more evidence of recombination. The observed patterns of the linkage disequilibrium statistic R2 suggest that a large number of sites will need to be genotyped to ensure coverage of the entire gene for genetic association studies. Identification of the sequence variation will allow genetic association studies of alpha8 in kidney and lung disease.     

Genomic organization and chromosome location of the murine Rpl23 gene

The mouse gene coding for ribosomal protein L23 (Rpl23) has been fully sequenced, including 580 bp of the 5' upstream region. The 5-kb gene comprises 5 exons and contains an unusually long (3,153 bp) third intron. The gene was mapped to the distal region of mouse chromosome 11, homologous to human chromosome 17q21-->q22.     

Genome organization and polymorphism of the murine beta-glucuronidase region

Thirty-eight kilobases of mouse genomic DNA which surround and include the coding sequences for beta-glucuronidase has been mapped. Intron-exon arrangements were determined by hybridization of genomic sequences with cDNA clones, and minimum estimates of gene length (11-17 kb) and intron number were obtained. Only a single gene was observed when genomic DNA was probed with subclones containing beta-glucuronidase coding sequence; there was no evidence of duplicated or pseudogenes. However, sequences distal to the 3' end of the gene are present elsewhere in the genome in a limited number of copies. Eight haplotypes of the beta-glucuronidase region with differing regulatory genotypes were compared for restriction fragment polymorphisms. Surprisingly little was found, considering the diverse origin of the haplotypes. Two of the polymorphisms that were found may be correlated with regulatory phenotypes. A BamHI site is missing from the CS and CL haplotypes that share regulatory properties, and a 0.2-kb insertion is consistently present in haplotypes showing increased response to induction by androgens in kidney.     

Genomic organization and chromosomal localization of the mouse protein kinase C alpha gene

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.     

Genetic organization of the polymorphic equine alpha globin locus and sequence of the BII alpha 1 gene.

The equine alpha globin gene complex comprises two functional alpha genes and an alpha-like pseudogene arranged in the order 5'-alpha 2-(5kb)-alpha 1-(3kb)-psi alpha-3'. A single (embryonic) zeta-like sequence lies within a 12 kb region 5' to the alpha 2 gene. We have determined the sequence of the alpha 1 gene of the BII haplotype, one of two most common haplotypes (the other being BI) which encode alpha globins with either Tyr (BI) or Phe (BII) at codon 24 in both linked alpha genes. In BI and BII the non-allelic alpha 2 and alpha 1 genes respectively code for Gln or Lys at codon 60, thus accounting for the 4 alpha globin types seen in BI/BII heterozygotes. Genomic restriction enzyme maps of the BII alpha complex (24Phe/60Lys,Gln) and the allelic BI (24Tyr/60Lys,Gln) are identical to each other, and to those of a rarer normal haplotype, A, which encodes only alpha 24Tyr/60Gln globin, and a low expression mutant of BII which encodes only 24Phe/60Lys globin. These two latter haplotypes must therefore have a linked pair of alpha genes, as in BI and BII, but with identical coding properties, and it is suggested that this has arisen by gene conversion.     

Genomic organization of the rat Clock gene and sequence analysis in inbred rat strains

While mutations in genes that function in the core molecular clock may disrupt circadian periodicity, their relevance to diurnal variation in metabolic, cardiovascular, and respiratory function is unknown. The circadian Clock gene product is an essential regulator of central and peripheral circadian rhythms in mammals. We have elucidated the complete exon-intron organization of the Clock gene in rat and have carried out an extensive search for single nucleotide polymorphisms (SNPs) in a panel of 12 inbred rat strains that exhibit diversity in studies of central and peripheral organ function and disease. The rat Clock gene consists of 23 exons spanning approximately 75 kb. Comparative sequence analysis identified 33 novel SNPs, including 32 that distinguish the Brown Norway (BN) rat from the other strains studied. Most notable were two novel mutations in the BN sequence at exon 8, Ile131Val and Ile132Val, occurring in a segment of the highly conserved PAS-A domain of the protein. These results afford the opportunity to assess the impact of genetic variation in Clock on central and peripheral functions subject to the core molecular clock and to test the importance of Clock variants in explaining diversity among rat strains in the expression of phenotypes, such as blood pressure, subject to circadian oscillation.