Genomic organization and chromosomal distribution of rat ID elements.

Identifier (ID) elements are members of a family of short interspersed nuclear elements (SINEs) in rodents. We investigated the genomic organization and chromosomal distribution of the ID elements in the rat, mouse and Chinese hamster. Southern blot hybridization analysis revealed that the ID elements are widespread in the rat genome, but concentrated in the mouse and Chinese hamster genomes, and that the copy of ID elements in the rat is about 5 times and 50 times that in the mouse and Chinese hamster, respectively. FISH analysis showed that the ID elements are predominantly distributed in the R-band regions of rat chromosomes. In mouse and Chinese hamster chromosomes, no specific distribution pattern of the ID elements was found. Furthermore, we found a distinct group of derivative ID elements in the rat, which contain partially repeated ID core domains, by PCR amplification using an ID core sequence. Such derivatives were not found in either the mouse or Chinese hamster. These findings suggest that explosive amplification of the ID elements in the rat has been accompanied by the occurrence of derivative ID elements and a predominant localization to the R-band regions. Similar associations found in the Alu family, one of the human SINEs, allow us to speculate that the rat ID elements and the human Alu family have analogous functions in chromosomal organization.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.     

The 5'region of the rat phosphoenolpyruvate carboxykinase gene confers pH sensitivity to chimeric genes expressed in renal and liver cell lines capable of expressing PEPCK

The 5' flanking regions of the rat phosphoenolpyruvate carboxykinase gene were used to form chimeric gene constructs with the human growth hormone gene. These constructs were transfected into several renal and one liver cell line and the production of growth hormone (HGH) measured by immunoassay. Cyclic-AMP and glucocorticoid responsiveness of HGH production was observed in all cell lines. In two lines, the rat NRK52E renal epithelial line and the rat H4IIE hepatoma cell line, both capable of expressing PEPCK, lowering extracellular pH increased HGH production several fold. Comparison of hormone and pH effect on cells transfected with a thymidine kinase promoter-HGH chimera indicated that the PEPCK 5' flanking region effects were specific. Thus, part of the pH responsiveness of the PEPCK gene in vivo may be attributed to properties of the 5' flanking regions.     

Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

Variations of elemental distribution in brain regions of neonatal rats at different iodine intakes

Iodine deficiency (ID) can result in irreversible damage to the brain during the fetal and neonatal stages. As the active center of many enzymes, trace elements play essential roles in brain function. In this work, the relative contents and distributions of elements (Cl, Br, Fe, Cu, and Zn) in two important brain regions, the cerebral cortex and hippocampus, of the iodine-deficient model rats were determined by the synchrotron radiation X-ray fluorescence (SRXRF) method. Meanwhile, the ID rats were supplemented with adequate and excessive iodine, respectively. The results indicated that the distributions of trace elements could be influenced by the different iodine intakes in the stage of brain development. In contrast to the control group, the contents of Cl, Br, and Zn in two brain regions showed a significant increase in the ID group; however, both Fe and Cu decreased in the cerebral cortex and increased in the hippocmapus in the ID group. In addition, only slight changes of elemental contents in brain were found between the adequate and excessive iodine-supplemented rats.     

Sex differences in structure and expression of the sex chromosome genes CHD1Z and CHD1W in zebra finches

Genes on the sex chromosomes are unique because of their sex-specific inheritance. One question is whether homologous gene pairs on the sex chromosomes, which have diverged in their sequence, have acquired different functions. We have analyzed the first homologous pair of genes (CHD1Z and CHD1W) discovered on the avian Z and W sex chromosomes of the zebra finch (Taeniopygia guttata) to examine whether functional differences may have evolved. Sequence analysis revealed that the two genes maintained a high degree of similarity especially within the C, H, and D domains, but outside of these regions larger differences were observed. Expression studies showed that CHD1W was unique to females and has the potential to produce a protein that CHD1Z does not. CHD1Z mRNA was expressed at a higher level in the male brain than in the female brain at various post-hatch ages. Reporter constructs containing the 5' flanking regions of each gene showed they had the ability to drive reporter expression in primary cell cultures. The 5' flanking region sequence of CHD1Z and CHD1W exhibited little homology, and differences in putative promoter elements were apparent. These differences between CHD1Z and CHD1W suggest that the two proteins may have diverged in their function.     

The rat interleukin-5 gene: characterization and expression by retroviral gene transfer and polymerase chain reaction.

The rat interleukin-5 (IL-5) gene was isolated from a genomic lambda phage library and a fragment containing all four exons was inserted into the retroviral vector pXT1, resulting in pXTRIL5. Upon retroviral gene transfer into two IL-5-dependent mouse cell lines, B13 and T88M, autonomously growing cells were established and B-cell growth factor activity was detected in the supernatants of the infected cells. "cDNA" versions of the rat IL-5 gene were rescued by the polymerase chain reaction (PCR) with primers specific for the flanking regions of the cloning site in pXT1. Restriction or DNA sequence analysis of five different clones revealed precise splicing in two cases, while three of the clones had retained the first intron. In addition, in two of these about 400 bp of rat IL-5 5' flanking regions were deleted. The sequence comparison of rat, mouse, and human IL-5 genes revealed a high degree of conservation (e.g., mouse and rat were 92% homologous at the amino acid level). The combination of retroviral gene transfer and PCR may offer an alternative, efficient method for the cloning of cDNAs.     

Organization and nucleotide sequence of the rat T cell receptor beta-chain complex.

We have characterized four overlapping genomic clones containing the DA rat TCR C beta complex, which span a total of 23 kb and bear two closely related complexes of gene segments. The D beta 1-J beta 1-C beta 1 and the D beta 2-J beta 2-C beta 2 complexes each contain a single diversity segment, six joining segments and four exons that encode the C region. All gene segments appear to be functional except J beta 2.5, which has a 5-bp frame-shifting deletion. This organizational pattern is identical to that of the mouse, and the homologous rat and mouse coding regions share about 92% nucleotide sequence identity. Our sequence comparisons indicate that a localized gene correction event has homogenized the sequences of the first exons of C beta 1 and C beta 2 in the evolutionary time since rats and mice became separate species. We have identified three repetitive elements, each flanked by short direct repeats, present in the region "brain-specific" identifier (ID) sequences, another is a truncated member of the LINE I class of repetitive elements, and the third is a member of the Alu type 2 family. The insertion of at least two, and probably all, of these elements has occurred since the time of rat/mouse divergence. We have identified a substantial number of "cryptic" rearrangement signals (heptamer/nonamer) in the C beta locus, which match the consensus sequence as well or better than authentic signals, and may represent sites of nonfunctional rearrangements.