Conserved sequences in both coding and 5'' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.     

The actin gene in yeast Saccharomyces cerevisiae: 5'' and 3'' end mapping, flanking and putative regulatory sequences

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.     

The 5'' flanking region of human epsilon-globin gene.

The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.     

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.     

Transfer RNA genes in Drosophila mitochondrial DNA: related 5'' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.     

Identification of the novel evolutionary conserved obstructor multigene family in invertebrates

Insects have evolved chitin-containing structures such as the cuticle or peritrophic membranes that serve to protect their bodies against the hostile environment. The specific mechanisms by which these structures are produced, are mostly unknown. We have identified a novel multigene family, the obstructor family, which encodes ten putatively secreted chitin-binding proteins that are characterized by a stereotype arrangement of a N-terminal signaling peptide and 3 chitin-binding-domains. Gene expression studies in Drosophila melanogaster embryos demonstrate that obstructor family members are expressed in cuticle forming tissues. Using computational and phylogenetic analysis, we show that obstructor genes represent an evolutionary conserved multigene family in invertebrates.     

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.     

Evolution of a multigene family of chorion proteins in silkmoths.

The evolution of the A family of chorion genes was examined by comparing new protein and DNA sequences from the silkmoths Antheraea pernyi and Bombyx mori with previously known sequences from Antheraea polyphemus. The comparisons indicated that the A family and its major subfamilies are ancient and revealed how parts of the genes corresponding to distinct regions of the protein structure have evolved, both by base substitutions and by segmental reduplications and deletions.