The Balbiani ring 2 gene in Chironomus tentans is built from two types of tandemly arranged major repeat units with a common evolutionary origin

The internal structure of the 37 kb long Balbiani ring 2 (BR 2) gene in Chironomus tentans has been studied by analysis of a collection of cloned cDNA sequences and in genomic Southern blot analysis with the cDNA sequences used as probes. The BR 2 gene contains two types of tandemly arranged major repeat units ˜200 bp long, represented in our study by the pCt 7 and the pCt 63 cDNA inserts. The pCt 7 major repeat units are arranged in one or possibly a few blocks and cover ˜10 kb of the gene; the pCt 63 units form one uninterrupted block, 22 kb in length. Genomic Southern blot hybridizations revealed a number of sequence variants of the pCt 7 major repeat unit. In contrast, the ˜100 copies of the pCt 63 major repeat unit seem to be almost identical. The pCt 7 major repeat unit, 180 bp in length, is organized in the same way as the previously described 215 bp long pCt 63 major repeat, i.e., it contains a repetitive and a non-repetitive part. Moreover, the two major repeat units show a high degree of sequence homology, indicating that the pCt 7 and pCt 63 sequence blocks within the Br 2 gene have evolved through stepwise amplification from a common ancestral sequence.     

A variant tandemly repeated nucleotide sequence in Balbiani ring 2 of Chironomus tentans

pCtBR2-2 is a genomic clone from Chironomus tentans that hybridized in situ to Balbiani ring 2 (BR2) on salivary gland polytene chromosome IV. DNA sequencing indicated that the insert contained nearly four copies of a 180 bp tandemly repeated nucleotide sequence that was distinctly different from a previously reported BR2 repeat. Sequence titration experiments detected about 70 copies of the 180 bp repeat per haploid genome, which would correspond to approximately 34% of a 37 kb BR2 gene. Each 180 bp repeat included a conserved 90 bp segment whose sequence was internally nonrepeating (INR), and a variable 90 bp repeated (R) segment comprised of three 30 bp repeats that may have evolved from a 9 bp consensus sequence. Results presented here raise the distinct possibility that other BR genes may contain significantly different repeated sequences that have not been identified.     

Structures of two HaeIII-type genes in the human salivary proline-rich protein multigene family

Two members of the human salivary proline-rich protein (PRP) multigene family have been isolated and completely sequenced. These PRP genes, PRH1 and PRH2, are of the HaeIII-type subfamily and code for acidic PRP proteins. Both genes are approximately 3.5 kilobase pairs (kb) in length and contain four exons. Exon 3 encodes the proline-rich part of the protein and includes five 63-base pair (bp) repeats. CAT and ATA boxes and several possible enhancer sequences occur in a 1-kb region 5' to exon 1. Two sets of repeats occur in the sequenced region in addition to the 63-bp repeats: one pair of about 140 bp flanks 500 bp of DNA in the first intervening sequence, and the other pair of 72 bp is tandemly repeated 1.4 kb 5' to the PRH1 gene. The 4-kb region of sequenced DNA from PRH1 differs by an average of 8.7% from the same region in PRH2, but the nucleotide sequences of the exon 3 of the two genes differ by only 0.2%. This result suggests the occurrence of a recent gene conversion event. The regions containing the 5-fold repeated sequences of 63 bp are identical in the two genes, PRH1 and PRH2. A comparison of the human HaeIII and BstNI subfamily repeats and a comparison of the human, mouse, and rat repeats suggest that the individual repeats have evolved in a concerted fashion within each gene and within the PRP gene family as a whole.     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences

We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.     

A family of long reiterated DNA sequences, one copy of which is next to the human beta globin gene.

An unusually long repeated DNA sequence was identified in cloned DNA, three kb 3' to the human beta-globin gene. Other members of this repeated sequence family were isolated from a human genomic DNA library and characterized by Southern blotting techniques, electron microscopy, and solution hybridization. The copy located next to the beta-globin gene was found to be 6.4 +/- 0.2 kb long and continuous over that length. This repeated sequence family comprises about 1% of the human genome and contains 3000-4800 copies of moderate sequence divergence which are interspersed with other less-highly repeated DNA. The 6.4 kb repeated unit does not appear to be composed of any smaller tandemly repeated subunits, nor is it expressed at a high level in bone marrow cell RNA.     

Characterization of the nicotinic acetylcholine receptor subunit gene Mdalpha2 from the house fly, Musca domestica

A nicotinic acetylcholine receptor (nAChR) subunit gene, Mdalpha2, was isolated and characterized from the house fly, Musca domestica. This is the first nAChR family member cloned from house flies. Mdalpha2 had a cDNA of 2,607 bp, which included a 696 bp 5'-untranslated region (UTR), an open reading frame of 1,692 bp, and a 219 bp 3'-UTR. Its deduced amino acid sequence possesses the typical characteristics of nAChRs. Mdalpha2 genomic sequence was 11.2 kb in length in the aabys strain and 10.9 kb in the OCR strain, including eight exons and seven introns. Based on the deduced amino acid sequence, Mdalpha2 had the closest phylogenetic relationship to the Drosophila melanogaster Dalpha2 and Anopheles gambiae Agamalpha2, and a similar genomic structure to Dalpha2. Quantitative real-time PCR analysis showed that Mdalpha2 is expressed in the head and the thorax at 150- and 8.5-fold higher levels than in the abdomen. Linkage analysis of a Mdalpha2 polymorphism indicates this gene is on autosome 2. The importance of these results in understanding the diversity and phylogenetic relationships of insect nAChRs, the physiology of nAChRs in the house fly, and the utility of nAChR sequences in resistance detection/monitoring is discussed.     

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

The gene of chlamysin, a marine invertebrate-type lysozyme, is organized similar to vertebrate but different from invertebrate chicken-type lysozyme genes

In a recent publication we reported the protein purification, characterization, and the gene isolation of a cDNA encoding the antibacterial cold-active lysozyme-like protein chlamysin from the marine bivalve Chlamys islandica. A 4.2 kb genomic chlamysin gene has now been amplified and sequence-analyzed. By comparison to the cDNA sequence and its translation product, the coding region was found separated in four exons of 38-252 bp. The introns range in size from 0.8 to 1.5 kb, and have traditional spliceosomal intron 5'-GT donor and 3'-AG acceptor sites for splicing. Two of the introns contain multiple copies of three sequence motifs not found repeated in other published genes. The over-all gene organization of chlamysin resembles chicken-type (c-type) lysozyme genes in vertebrates, but is different from the three-exon structure in invertebrate c-type lysozyme genes. A phylogenetic analysis of invertebrate-type (i-type) and c-type lysozyme proteins demonstrated a large evolutionary distance between the i-type and the c-type enzyme classes. Exons of the i-type genes are not equally organized according to their homolog protein domains.     

Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome.

Sixty-five TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of Lotus japonicus accession MG-20 based on the sequence information of expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The average insert size of the TAC clone was approximately 100 kb, and the total length of the sequenced regions in this study is 6,556,100 bp. Together with the nucleotide sequences of 56 TAC clones previously reported, the regions sequenced so far total 12,029,295 bp. By comparison with the sequences in protein and EST databases and by analysis with computer programs for gene modeling, a total of 711 potential protein-encoding genes with known or predicted functions, 239 gene segments and 90 pseudogenes were identified in the newly sequenced regions. The average gene density assigned so far was 1 gene/9140 bp. The average length of the assigned genes was 2.6 kb, which is considerably larger than that assigned in the Arabidopsis thaliana genome (1.9 kb for 6451 genes). Introns were identified in approximately 73% of the potential genes, and the average number and length of the introns per gene were 3.4 and 377 bp, respectively. Simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated based on the nucleotide sequences of the genomic clones obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

A BR 1 gene in Chironomus tentans has a composite structure: a large repetitive core block is separated from a short unrelated 3''-terminal domain by a small intron.

The large Balbiani ring (BR) genes in the dipteran genus Chironomus have been considered to be homogeneous repetitive structures. Analysis of a genomic DNA segment now reveals that a BR 1 gene in C. tentans is a composite gene, consisting of two different types of sequences. A 15-20 kb core block of tandemly arranged repeat units extends close to the 3' end of the BR 1 gene and ends in repetitive structures partly different from the repeat units in the core block. A 55 bp long intron separates the core block, which probably constitutes a single exon, from a non-related 3'-exon, comprising the final 332 bp of the translated part of the gene. According to hydrophobicity and secondary structure predictions, the 3'-exon encoded peptide is distinctly different from the repetitive core block domain and attains a globular structure. The carboxyl-terminal peptide domain is likely to be a general feature of BR encoded proteins and may have important functions in the excretion and polymerisation of the secretory proteins.     

Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice.

The serum level of the fourth component of complement (C4) in mice bearing the H-2k haplotype is only 1/10 to 1/20 of that of non-H-2k mice. We have analyzed C4 cDNA clones from B10.BR(H-2k) mouse liver and found aberrant C4 cDNA which contained a 200-base pair (bp) insertion between the exon 13 and exon 14 encoded sequences in addition to the normal C4 cDNA. The 5' 148 bp and the 3' 52 bp of this insert were derived from the B2 sequence, the short interspersed repeats of mouse genome, and the central part of intron 13, respectively. Sequence analysis of intron 13 of the C4k gene showed the presence of a complete copy of a B2 consensus sequence. The structure of aberrant C4 mRNA indicated that the possible 3' splice site in the B2 sequence and the cryptic 5' splice site in intron 13 were used. Both the insertion of the B2 sequence into intron 13 and the presence of aberrant mRNA in the liver were specific to H-2k-bearing mice, suggesting that the aberrant splicing due to the B2 insertion is the basis for low C4 expression in H-2k mice.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Molecular cloning of LMO41, a new human LIM domain gene

We have identified a new human LIM domain gene by isolating an autoantigenic cDNA clone from a human breast tumor cDNA library. The predicted amino acid sequence of the cDNA clone's 495 bp open reading frame contains two tandem LIM domain motifs, and within the LIM domain region there is 62% identity with the analogous region of the LIM-only gene LMO1. The homology to LMO1 is restricted to the 360 bp region encoding the tandemly repeated LIM domains, the rest of the open reading frame as well as the extensive, GC-rich 5' untranslated region, and 3' region of the 2 kb cDNA sequence are unrelated to any known genes. This gene has been designated LMO4.