The alpha1-microglobulin/bikunin gene: characterization in mouse and evolution.

The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.     

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

Molecular biology of neurohormone precursors in the eyestalk of Crustacea

Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.     

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host-parasite adaptation.     

The structure of precursor mRNAs and of excised intron RNAs in chloroplasts of Euglena gracilis

Partially spliced precursor mRNAs (pre-mRNAs) in the steady-state population of RNA from chloroplasts of Euglena gracilis were found by electron microscopy. The structure and the frequency of the pre-mRNAs of the psbA gene (the gene for the 32-kd protein of photosystem II), which is split by four introns in Euglena chloroplasts was analysed by electron microscopy. A chloroplast DNA (cpDNA) fragment containing the psbA gene from Euglena, was cloned into a pEMBL vector. The single-stranded recombinant phage DNA of the coding strand was prepared and hybridized with cpRNA. The majority of hybrids were formed with mature mRNA, but approximately 8% of the hybrids were formed with pre-mRNAs. The pre-mRNAs were either unspliced or incompletely spliced. A detailed analysis of the structure and the frequency of the pre-mRNAs of the psbA gene showed that the four introns are neither spliced out in a strictly random way, nor in a 5'-3' or 3'-5' direction. Introns 2 and 3 are preferentially spliced out first, intron 1 intermediately and intron 4 is generally spliced out last. However, this sequence is not a strict rule. We conclude that the introns can be spliced independently, each one at a different rate. The coding strand from a fragment of the psbA gene was separated and annealed with low mol. wt. cpRNA, which was isolated from an agarose gel. Small circular hybrids were found at the positions of the four introns, demonstrating for the first time covalently closed circular excised intron RNAs (iRNAs) in chloroplasts.     

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.     

Molecular cloning of the human thyrotropin-beta subunit gene

Genomic DNA fragments that carried a gene for human thyrotropin-beta (hTSH beta) subunit were isolated. Nucleotide sequence analysis of the gene showed that the hTSH beta subunit precursor consists of 138 amino acid residues. There is an N-terminal sequence of 20 amino acids as a signal peptide, followed by 112 amino acids, whose sequence is in agreement with that known for the secretory form of hTSH beta subunit. This is followed by an additional stretch of 6 hydrophobic amino acids, which may be eliminated post-translationally. The coding region is separated by an intron of about 460 bp. Genomic Southern blot hybridization analysis suggested that the hTSH beta gene is a unique single copy gene.     

Sequence and methylation in the beta/A4 region of the rabbit amyloid precursor protein gene.

Alzheimer's disease is characterized by the accumulation of the beta/A4 fragment of the amyloid precursor protein in the hippocampal regions of the brain. We report here the isolation of genomic clones carrying exons 15, 16 and 17 of the beta/A4 coding region of the rabbit amyloid precursor protein gene. The complete sequence of these exons predicts that all three peptides are identical to their human counterparts. An unexpectedly high concentration of CpG dinucleotides seen in exon 15 were conserved and continued into the intron 15 region. MspI/HpaII southern blot analysis revealed the presence of a number of methylated CpG dinucleotides in the cloned region of the gene. These data suggest that the rabbit amyloid precursor protein gene could provide a new and useful model for the study of this important gene.     

The PreA4(695) precursor protein of Alzheimer''s disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.     

Mutational analysis of the amyloid precursor protein gene in Japanese familial Alzheimer's disease kindreds

We sequenced the entire coding region of the amyloid precursor protein (APP) genes of 11 unrelated patients with Japanese familial Alzheimer's disease (FAD) in order to determine the exact frequency of known APP gene mutations and to search for novel mutations responsible for FAD. Three out of 11 (27.3%) FAD patients showed the known Val to Ile mis-sense mutation at codon 717, but no other mutations were detected in the entire coding region. Analysis of exons 16 and 17 in 30 Japanese with sporadic AD revealed no mutations. Moreover, there were no significant differences in the allele frequencies of the DNA polymorphism in intron 9 among the 11 FAD, 39 sporadic AD, and 110 control subjects.     

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host–parasite adaptation.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

The NADH-dehydrogenase subunit 5 gene in Oenothera mitochondria contains two introns and is co-transcribed with the 5 S rRNA gene

Summary The gene encoding subunit 5 of the NADH dehydrogenase complex (ND 5) has been identified in Oenothera mitochondria from a cDNA clone. The coding region is interrupted by a type II intron of 850 nucleotides and a second intervening sequence of 357 nucleotides. Genomic sequence rearrangement within the first intron creates a nontranscribed partial copy of the gene. The intact ND 5 gene is transcribed in a complex pattern with mRNAs including the 5 S rRNA sequence. Excision of the two introns appears to proceed slowly in vivo since the steady state mitochondrial RNA contains significant proportions of unprocessed precursor molecules.