The Saccharomyces cerevisiae MET3 gene: nucleotide sequence and relationship of the 5'' non-coding region to that of MET25

Summary In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is coregulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5 and 3 flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250–280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.     

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

Summary The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5 flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3 end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).     

High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions

The 1.4 kb 5 polygalacturonase (PG) gene-flanking region has previously been demonstrated to direct ripening-specific chloramphenicol acetyl transferase (CAT) expression in transgenic tomato plants. The steady state level of CAT mRNA in these plants was estimated to be less than 1% of the endogenous PG mRNA. Further constructs containing larger PG gene-flanking regions were generated and tested for their ability to direct higher levels of reporter gene expression. A 4.8 kb 5-flanking region greatly increased levels of ripening-specific reporter gene activity, while a 1.8 kb 3 region was only shown to have a positive regulatory role in the presence of the extended 5 region. Transgenic plants containing the CAT gene flanked by both of these regions showed the same temporal pattern of accumulation of CAT and PG mRNA, and steady-state levels of the transgene mRNA were equivalent to 60% of the endogenous PG mRNA on a per gene basis. The proximal 150 bp of the PG promoter gave no detectable CAT activity. However, the distal 3.4 kb of the 4.8 kb 5 PG promoter was shown to confer high levels of ripening-specific gene expression when placed in either orientation upstream of the 150 bp minimal promoter. The DNA sequence of the 3.4 kb region revealed a 400 bp imperfect reverse repeat, and sequences which showed similarity to functionally significant sequences from the ripening-related, ethylene-regulated tomato E8 and E4 gene promoters. The possible roles of the flanking regions in regulating PG gene expression are discussed.     

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

Cloning of the fibroin gene from the oak silkworm, Antheraea yamamai and its complete sequence

The nucleotide sequences containing an entire genomic region and 5 upstream region of Antheraea yamamai fibroin gene have been determined. The gene consists of an initial exon encoding 14 amino acids, an intron (150 bp), and a long second exon coding for 2641 amino acids. The fibroin coding sequence shows a specialized organization with a highly repetitive region flanked by non repetitive 5 and 3 ends. Northern blot analyses confirmed that fibroin gene is actively expressed in the posterior silk gland of the final instar larvae of Antheraea yamamai.     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

The L1 family of long interspersed repetitive DNA in rabbits: Sequence,copy number,conserved open reading frames,and similarity to keratin

Summary The L1 family of long interspersed repetitive DNA in the rabbit genome (L1Oc) has been studied by determining the sequence of the five L1 repeats in the rabbit -like globin gene cluster and by hybridization analysis of other L1 repeats in the genome. L1Oc repeats have a common 3 end that terminates in a poly A addition signal and an A-rich tract, but individual repeats have different 5 ends, indicating a polar truncation from the 5 end during their synthesis or propagation. As a result of the polar truncations, the 5 end of L1Oc is present in about 11,000 copies per haploid genome, whereas the 3 end is present in at least 66,000 copies per haploid genome. One type of L1Oc repeat has internal direct repeats of 78 bp in the 3 untranslated region, whereas other L1Oc repeats have only one copy of this sequence. The longest repeat sequenced, L1Oc5, is 6.5 kb long, and genomic blot-hybridization data using probes from the 5 end of L1Oc5 indicate that a full length L1Oc repeat is about 7.5 kb long, extending about 1 kb 5 to the sequenced region. The L1Oc5 sequence has long open reading frames (ORFs) that correspond to ORF-1 and ORF-2 described in the mouse L1 sequence. In contrast to the overlapping reading frames seen for mouse L1, ORF-1 and ORF-2 are in the same reading frame in rabbit and human L1s, resulting in a discistronic structure. The region between the likely stop codon for ORF-1 and the proposed start codon for ORF-2 is not conserved in interspecies comparisons, which is further evidence that this short region does not encode part of a protein. ORF-1 appears to be a hybrid of sequences, of which the 3 half is unique to and conserved in mammalian L1 repeats. The 5 half of ORF-1 is not conserved between mammalian L1 repeats, but this segment of L1Oc is related significantly to type II cytoskeletal keratin.     

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours