Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori

Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for the protein, we determined the primary structures of its partial peptides, and the gene was searched using the silkworm genomic databases. Three contigs containing the corresponding nucleotide sequences were identified and categorized as one group. The gene structure covering the 5' flanking and the 3' end was determined by PCR fragments from genomic DNA, RT-PCR, and 5' and 3' RACE. The amino acid sequence deduced from the nucleotide sequence mainly consists of two serine-rich regions of 86-amino acid motif and 8-amino acid repeated sequence. The expression of the gene is limited to the anterior and middle parts of the middle silk gland. In addition, because the sericin gene appeared different from the sericin 1 and 2 genes reported earlier, we designated the newly discovered gene as sericin 3.     

Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation.

The nucleotide sequence of the parvovirus H-1 has been determined by the chain-terminating method of Sanger. The sequence is 5,176 nucleotides long. Two large open reading frames (1 and 2) and two smaller open reading frames (3 and 4) of potential importance were identified in the plus-strand sequence. Promoter sequences are located at map positions 4 and 38 when map positions are expressed as percent of genome length from the 3' end of the virion minus strand. The locations for the genes for the parvovirus capsid proteins and a 76,000-dalton noncapsid protein (NCVP1) were mapped by hybrid-arrested translation. The gene for the capsid proteins VP1 and VP2' is located in the 5' half of the virus genome. The gene for NCVP1 is located in the 3' half of the viral DNA.     

Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A.

A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.     

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.     

Structure of a cluster of mouse histone genes.

The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes.     

Expression of a tuber-specific storage protein in transgenic tobacco plants: demonstration of an esterase activity

A chimaeric gene composed of the 5' upstream region of STLS1, a leaf/stem specifically expressed gene from Solanum tuberosum, and the RNA-coding as well as the 3' downstream region of patatin, the major storage protein of potato tubers, has been transferred into tobacco plants using the Agrobacterium system. The introduction of this gene led to a leaf/stem specific expression of a 42-kd large protein which immunocrossreacts with patatin antiserum. Only low amounts of immunoreacting protein of smaller size could be detected in transgenic tobacco leaves indicating that the patatin protein is fairly stable in this heterologous environment. The size of the protein as well as the size of the RNA detected in transgenic tobacco leaves using a patatin-specific probe indicates that the patatin RNA was accurately processed in both leaf and stem tissue of tobacco. The expression of the patatin gene led to the appearance of a new esterase activity in the transformed tobacco which co-migrated with a protein immunoreacting with patatin antiserum. These data therefore demonstrate that patatin in addition to serving as a storage protein displays an enzymatic activity.     

Sequence homologies within the 5'' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken.

In oviparous vertebrates vitellogenin, the precursor of the major yolk proteins, is synthesized in the liver of mature females under the control of estrogen. We have established the organization and primary structure of the 5' end region of the Xenopus laevis vitellogenin A2 gene and of the major chicken vitellogenin gene. The first three homologous exons have exactly the same length in both species, namely 53, 21 and 152 nucleotides, and present an overall sequence homology of 60%. In both species, the 5'-non-coding region of the vitellogenin mRNA measures only 13 nucleotides, nine of which are conserved. In contrast, the corresponding introns of the Xenopus and the chicken vitellogenin gene show no significant sequence homology. Within the 500 nucleotides preceding the 5' end of the genes, at least six blocks with sequence homologies of greater than 70% were detected. It remains to be demonstrated which of these conserved sequences, if any, are involved in the hormone-regulated expression of the vitellogenin genes.     

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5'' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences.

The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein. The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined. The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA. The DNA and protein sequences established that the mature protein is not processed from a precursor form. No sequence corresponding to an SOS box was found in the 5' sequence of DNA. There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding. The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene. A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences.