Expression of Gtf2ird1, the Williams syndrome-associated gene, during mouse development

The gene GTF2IRD1 is localized within the critical region on chromosome 7 that is deleted in Williams syndrome patients. Genotype-phenotype comparisons of patients carrying variable deletions within this region have implicated GTF2IRD1 and a closely related homolog, GTF2I, as prime candidates for the causation of the principal symptoms of Williams syndrome. We have generated mice with an nls-LacZ knockin mutation of the Gtf2ird1 allele to study its functional role and examine its expression profile. In adults, expression is most prominent in neurons of the central and peripheral nervous system, the retina of the eye, the olfactory epithelium, the spiral ganglion of the cochlea, brown fat adipocytes and to a lesser degree myocytes of the heart and smooth muscle. During development, a dynamic pattern of expression is found predominantly in musculoskeletal tissues, the pituitary, craniofacial tissues, the eyes and tooth buds. Expression of Gtf2ird1 in these tissues correlates with the manifestation of some of the clinical features of Williams syndrome.     

A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression

Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.     

Fiber-FISH analysis of the 3'-terminal region of the human L-type Ca2+ channel alpha 1C subunit gene

Human L-type Ca2+ channel alpha 1C subunit gene (CACNL1A1) maps to the distal region of chromosome 12p13, and is composed of approximately 50 exons spanning over 150 kb of the human genome as estimated by restriction map analysis. However, the structure and the total length of the 3'-end of the gene is not clear because the size of several big introns remains unknown. Here the fiber-FISH technique was used to determine the relative order and size of eight partial genomic DNA clones from the central and 3'-terminal regions of CACNL1A1. The total physical distance of this region, including the size and gap distances between the clones were re-estimated. The results show that the physical order of the tested clones was 5'-g14-5 > g12-2 > g10-8 > g4-5 > g16-7 > g8-3 > g12-5 > g6-20-3'. Their individual sizes vary between 6.7 and 21.9 kb. Clones g6-20 and g12-5, both containing repetitive exon 45/46-like element, were found to be located within 59.1 kb downstream of g8-3 containing earlier identified polyadenylation site, i.e. 229.5 kb away from clone g14-5 (exons 10, 11). The possible implications of this structural complexity is discussed.     

Evolutionary conservation of the apolipoprotein E-C1-C2 gene cluster on bovine chromosome 18q24

We have constructed a long-range restriction map spanning about 250 kb on bovine chromosome 18q24. Our results show that the apolipoprotein C2 (APOC2) gene is located about 25 kb from the APOE gene. Four putative CpG islands are also indicated in the map. Interestingly, a minisatellite located in the third intron of the human and mouse APOC2 genes was also found at identical position in the bovine gene and revealed high sequence identity comparing with the two corresponding sequences. By means of cosmid mapping, we further demonstrate that the APOE-APOC1-APOC2 gene cluster is evolutionary conserved in cattle.