Organization of the human interleukin-1 receptor antagonist gene IL1HY1

 The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7600 nucleotides and found four coding exons ranging in size from 55 to 2288 nucleotides. The 5′ untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene. Received: 15 October 1999 / Revised: 30 December 1999     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Divergent intron arrangement in theMB1/LMP7 proteasome gene pair

We sequenced the humanMB1 gene from a cosmid clone mapping to chromosome 14q11.2–12. The gene spans about 6 kilobases and contains three exons and two introns. There was no evidence of an alternative leader exon, which is a characteristic of the major histocompatibility complex (MHC)-encodedLMP7 gene, the closet relative ofMB1, with which it shares 67% amino acid identity. Conceptual translation of the 5′ end of the gene class for a cleaved leader sequence of 59 amino acids, consistent with western blot data. None of theMB1 gene's three exons were coincident with any of the six exons inLMP7. In contrast, in the delta-encoding gene and its counterpart, the MHC-encodedLMP2 gene (59% amino acid identity), all six exons are arranged at equivalent positions in respect to the coding frame. The unique structure ofMB1 implies a separate origin or different selection pressures acting at this particular locus. DNA repeat analysis provides information on the minimum time of separation of theMB1/LMP7 pair of genes. The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X95586     

Repetin (Rptn), a New Member of the “Fused Gene” Subgroup within the S100 Gene Family Encoding a Murine Epidermal Differentiation Protein

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pIof 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescencein situhybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the “fused gene” subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.     

Characterization of the cDNA and Gene Encoding Human PDE3B, the cGIP1 Isoform of the Human Cyclic GMP-Inhibited Cyclic Nucleotide Phosphodiesterase Family

Two distinct PDE3 [cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been identified. Here we report cloning of the cDNA and gene encoding human (H)cGIP1 (classified as PDE3B). The cDNA encodes a protein of 1112 amino acids (123 kDa). Northern blots indicate that its mRNA is expressed in several adipose tissue depots. The human PDE3B gene is composed of 16 exons spanning more than 114 kb and was localized to chromosome 11p15 byin situhybridization. Exon/intron boundaries were determined, and genetic polymorphism, confirmed by single-strand conformational polymorphism of DNA from 25 healthy subjects, was demonstrated in exon 4 at nucleotide 1389 (A/G). Two polymorphic dinucleotide repeat sequences were identified in introns 5 and 12.     

Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH₂-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.     

Nucleotide sequence of the gene for the b subunit of human factor XIII

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.     

Cloning of the human RoDH-related short chain dehydrogenase gene and analysis of its structure

We have previously characterized the first human NAD⁺-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5′-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3α-hydroxysteroid dehydrogenase (3α-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3α-HSD has four translated exons and, possibly, a fifth exon that codes for the 5′-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon–intron boundaries and the sizes of the protein coding regions are identical in 3α-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3α-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.     

The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene.

The ALL-1 gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 4, 6, 9, 10, or 19. The gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of more than 3910 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene, including cysteine-rich regions that can be folded into six zinc finger-like domains. The breakpoint cluster region within ALL-1 spans 8 kb and encompasses several small exons, most of which begin in the same phase of the open reading frame. The t(4;11) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-1 and from a gene on chromosome 4. This suggests that each 11q23 abnormality gives rise to a specific oncogenic fusion protein.     

Cloning of the full-length coding sequence of rat liver-specific organic anion transporter-1 (rlst-1) and a splice variant and partial characterization of the rat lst-1 gene

The full-length coding sequence of rat liver-specific organic anion transporter-1 (lst-1) and its splice variant have been cloned. The full-length rat lst-1 (designated rlst-1a) encodes a protein containing 687 amino acids and has 12-putative transmembrane domains, multiple potential N-glycosylation and phosphorylation sites. Therefore, rat lst-1a has 35 additional amino acid residues compared to the previously reported rat lst-1. A splice variant (designated rlst-1c) reported in this communication encodes a protein containing 654 amino acids and has 10-putative transmembrane domains. PCR analysis suggests that rlst-1a is the most abundant form in liver. Phylogenetic analysis reveals that rat lst-1a is an ortholog of human LST-1 (hLST-1) and mouse lst-1 (mlst-1). The rlst-1 gene is composed of 15 exons and 14 introns. Analysis of exon-intron boundary reveals that the splice variant rlst-1c lacks the entire exon 7, while the previously reported rat lst-1 (designated herein as rlst-1b) lacks approximately half of exon 10, and the splicing has occurred through alternative usage of a splice donor site within exon 10.