The histone H5 gene is flanked by S1 hypersensitive structures.

The potential of the cloned histone H5 gene to form altered DNA structures has been examined by S1 nuclease digestion of supercoiled recombinant plasmids containing up to 8.8 kbp of chicken DNa. The three main nicking sites map at the upstream and downstream sequences flanking the structural gene. The cleavage sites share sequence homology, strand specificity, and do not seem to be single-stranded. The sequence of the S1-sensitive sites does not suggest that the fragments can adopt any of the known DNA secondary structures.     

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.     

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.     

Fugu immunoglobulin D: a highly unusual gene with unprecedented duplications in its constant region

We have isolated and characterized the cDNA that encodes IgD of fugu (Takifugu rubripes). Though the splicing of 1 with the 1 domain was similar to those reported for teleost IgDs, highly unusual and unprecedented domain duplications were found in the constant region of the fugu IgD. The structure of the fugu IgD is like VDJ-1-(1-2-3-4-5-6)₂-7-m1-m2. Genomic sequence analysis of the fugu IgD gene supported the results of cDNA sequencing that the first six domains in the constant region are duplicated. Such a novel duplication pattern has not been reported in any other vertebrates. However, IgD secretory domains could not be identified in this study. The deduced amino acid sequence of the fugu IgD constant region showed high identity (35–55%) to the sequences of previously reported teleost IgDs. Gene expression analyses based on RT-PCR demonstrated that the IgD gene is preferentially expressed in presumptive lymphoid tissues; moreover, in situ hybridization showed that IgD-positive cells are distributed throughout the spleen and head kidney. The expression pattern is similar to that of IgM, corroborating the hypothesis that IgD plays an important role in the humoral immune system of this species.The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB159481 and AB159482.     

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.     

Aspartyl aminopeptidase, encoded by an evolutionarily conserved syntenic gene, is colocalized with its cluster in secretory granules of pancreatic islet cells

Aspartyl aminopeptidase (DAP), encoded by the DNPEP gene, is believed to be a cytosolic protein with high enzymatic activity in the neuroendocrine tissues. Bioinformatic analysis revealed that the genomic segment spanning the DNPEP gene is evolutionarily conserved from Caenorhabditis elegans to humans. In the present study, we sought to determine whether the expression of DAP is associated with its clustered genes when expressed in pancreatic islet cells. Using anti-DAP specific antibody in immunofluorescent stainings, we found that DAP was specifically expressed in islet alpha cells but not in exocrine acinar cells. Moreover, using electron microscopy, we found that DAP was associated with a lysosomal-like structure and secretory granules, suggesting that it plays an important role in post-translational processing and the secretion of hormones in islet cells. The identification and characterization of DNPEP syntenic genes confirm that conserved clustered genes can preferentially be expressed in the same signaling pathway.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

Mouse melanoma growth stimulatory activity gene (Mgsa) is polymorphic and syntenic with the W, patch, rumpwhite, and recessive spotting loci on chromosome 5

Melanoma growth stimulatory activity (Mgsa) is a polypeptide growth factor originally detected in culture medium of the human malignant melanoma cell line Hs294T and may have an autocrine role in neoplastic growth. Mgsa is a member of the small inducible gene (SIG) family and shares homology with beta-thromboglobulin and platelet factor 4. Mgsa was localized to chromosome 5 using a cDNA probe for mouse Mgsa and somatic cell hybrids and is thus syntenic with Kit (W), Ph, Rw, and rs loci. The results eliminate Mgsa as the product of the Steel locus on chromosome 10, but raise the possibility that Mgsa might be synonymous with a chromosome 5 locus affecting skin pigmentation.     

Mouse ferritin H multigene family is polymorphic and contains a single multiallelic functional gene located on chromosome 19.

Multiple ferritin H subunit sequences are present in the genome of higher vertebrates, but it is not yet known with certainty if more than one is expressed. In this paper, we provide evidence that there is only one functional ferritin H gene in the mouse. We screened a mouse genomic library using a mouse ferritin H cDNA as a probe and characterized five clones. These genomic clones proved to contain three pseudogenes and two allelic forms of a unique functional gene. These two alleles differed by only two point mutations in the promoter and three in the first intron and by a 31-bp insertion in the first intron. They were equally expressed when transiently transfected in HeLa cells. These five genomic clones account for all the bands observed on a Southern blot of mouse genomic DNA hybridized with a ferritin H cDNA, and these bands present a restriction fragment length polymorphism between various representatives of the genus Mus. Using a DNA panel prepared from the backcross progeny (C57BL/6 X Mus spretus)F1 X C57BL/6, we localized the functional ferritin H gene (Fth) in region B of mouse chromosome 19 and established cen-Ly-1-Fth-Pax-2 as the most likely gene order, thus defining a conserved syntenic fragment with human chromosome 11q.     

Identification of a processed pseudogene related to the functional gene encoding the GM2 activator protein: localization of the pseudogene to human chromosome 3 and the functional gene to human chromosome 5.

The GM2 activator protein is an essential substrate cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A (EC 3.2.1.52). There have been conflicting reports as to the chromosomal localization of the gene encoding the activator. We demonstrate here that these conflicts were caused by the presence of a previously unidentified processed activator-pseudogene on chromosome 3, and we confirm a previous ELISA-based localization of the functional activator gene to chromosome 5. Our data indicate that the functional activator locus can still be considered a candidate site for defects causing some forms of spinal muscular atrophy.     

CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2

Background

Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation) and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD) event (ohnologs) versus small-scale duplications (SSD) to determine if there exist any differences in their patterns of sequence evolution.

Results

For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like) in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression.

Conclusions

Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.     

Complete structure of the human transferrin gene. Comparison with analogous chicken gene and human pseudogene

The complete structure of the human transferrin gene is presented. This gene has a total size of about 33.5 kb and is organized in 17 exons separated by 16 introns. The chicken ovotransferrin gene has a size of 10.5 kb and is also organized in 17 exons and 16 introns. The analysis of the structure of the two genes confirm, at the gene level, that transferrins originated by a gene duplication phenomenon. Finally, the existence of a new member of the transferrin family, a human transferrin non-processed pseudogene is demonstrated.     

Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization

Protein S is a vitamin K dependent plasma protein and a cofactor to activated protein C, a serine protease that regulates blood coagulation. The haploid genome contains two protein S genes (alpha and beta) with the protein S alpha-gene corresponding to the cloned cDNA. We have now isolated and mapped overlapping genomic clones that cover an area of 50 kilobases of the protein S alpha-gene which code for the 3' part of the gene, i.e., the thrombin-sensitive region, the four domains that are homologous to the epidermal growth factor (EGF) precursor, the COOH-terminal part of protein S that is homologous to a plasma sex hormone binding globulin (SHBG), and, finally, the 3' untranslated region. The thrombin-sensitive region and the EGF-like domains are each coded on a separate exon. The sizes of the exons coding for the COOH-terminal half of protein S and the location of the introns are nearly identical with those in the homologous SHBG gene. Furthermore, the phase class of the splice junctions is the same in these two genes. We have also isolated and mapped genomic clones that cover 25 kilobases of the protein S beta-gene, which was found to contain stop codons and a 2 bp deletion which introduces a frame shift, suggesting that it is a pseudogene. The structure of the two protein S genes and a comparison with the vitamin K dependent clotting factors support a model for their origin by exon shuffling and recruitment of the 3' part of the gene from an ancestor shared with the sex hormone binding globulin.     

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.     

Amino acid change associated with the major polymorphic Hinc II site of Oriental and Caucasian mitochondrial DNAs.

The mitochondrial DNAs (mtDNAs) from 116 Oriental and Caucasian blood samples were analyzed for their Hinc II restriction endonuclease cleavage patterns using Southern analysis and 32P human mtDNA probes. Seven distinct patterns were found, all of which could be interrelated by single nucleotide changes. The predominant pattern (mtHinc II-2) was found in 97% of the Caucasians and 73% of the Orientals. This mtDNA morph had one more Hinc II site than did the second most common morph (mtHinc II-1), which was found only in 20% of the Orientals. Three additional patterns were in a single Oriental sample, a fourth in a single Caucasian sample, and a fifth in one member of each population. The polymorphic site that differentiated mtHinc II-1 and mtHinc II-2 was cloned and sequenced. A single nucleotide change was found that created an Hinc II site and changed the amino acid sequence of the URF5 gene. Comparison of these sequences with those of other primates [15] revealed that the Asian mtHinc II-1 and mtHinc II-4 mtDNAs were identical in this region with those of chimpanzees and orangutans. These results suggest that the Asian mtHinc II-1 mtDNA may have been ancestral to other human mtDNAs.     

Identification of a single missense mutation in the adenine phosphoribosyltransferase (APRT) gene from five Icelandic patients and a British patient.

We have completely sequenced the adenine phosphoribosyltransferase (APRT) gene from each of six patients--five (I-V) from Iceland and one (VI) from Britain. Cases I and II shared a common ancestor six and seven generations ago, and cases I and V shared a common ancestor seven generations ago, but cases III and IV were unrelated to the above or to each other, over seven generations. Genomic DNA was amplified by PCR, subcloned into M13mp18, and sequenced. Genomic and PCR-amplified DNAs were also analyzed by restriction-enzyme digestion and Southern blotting. The same missense mutation was identified in all six patients. This mutation leads to the replacement of asp (GAC) by val (GTC), at amino acid position 65. The gene sequences from all patients were otherwise identical to our wild-type sequence. The homozygous nature of the mutation was confirmed by sequencing the PCR product directly. All six patients were homozygous for the 1.25-kb TaqI RFLP. The Icelandic patients were also homozygous for the 8-kb SphI RFLP, but the British patient was heterozygous at this site. These studies suggest that a founder effect is likely to be responsible for APRT deficiency in the Icelandic population. The finding of the same mutation in a patient from Britain suggests that this mutation may have originated in mainland Europe.