Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses.

Circoviruses are a diverse group of animal and plant pathogens with undefined relationships to one another but for their non-geminate, non-enveloped capsids and circular, single-stranded DNA genomes. The sequences of the beak and feather disease virus and porcine circovirus genomic DNAs are presented and analyzed in the context of the other members of the family. Sequence comparisons, inferred phylogenies, and geographic occurrence suggest that the ambisense circoviruses, particularly the beak and feather disease virus, represent an evolutionary link between the geminiviruses and the plant circoviruses. We propose that the family members be reclassified into three groups: The family Circoviridae consists of the animal pathogens (beak and feather disease virus and porcine circovirus) that possess ambisense genomes with striking similarities to the geminiviruses. The BBTV-like viruses include the plant pathogens (coconut foliar decay virus, banana bunchy top virus, subterranean clover stunt virus) with a geminivirus-like stem-loop element in their DNAs, and single to multiple component genomes. The chicken anemia virus is an unassigned virus possessing unique characteristics bearing little similarity to the other ssDNA viruses.

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions.

Five of the genes for the biosynthesis of isoleucine and valine form the ilvGMEDA operon of Escherichia coli K-12. Expression of the operon responds to changes in the availability of isoleucine, leucine, and valine (ILV). Addition of an excess of all three amino acids results in reduced expression of the operon, whereas limitation for one of the three amino acids causes an increase in expression. The operon is preceded by a leader-attenuator which clearly regulates the increased expression that occurs due to reduced aminoacylation of tRNA. To assess the factors that result in the reduced expression of this operon upon the addition of ILV, a series of plasmids were constructed in which the ilv regulatory region was fused to galK. In response to addition of the amino acids, expression of the galK gene fused to the leader-attenuator decreased five- to sevenfold, instead of the twofold observed for the chromosomal operon. A deletion analysis with these plasmids indicated that the ILV-specific decrease in expression required an intact leader-attenuator but not ilvGp2 or the DNA that precedes this promoter. This conclusion was supported by both S1 nuclease analysis of transcription initiation and determination of galK mRNA levels by RNA-RNA hybridization.

Transcriptional polarity enhances the contribution of the internal promoter, ilvEp, in the expression of the ilvGMEDA operon in wild-type Escherichia coli K12.

The ilvG gene of Escherichia coli K12 produces a cryptic peptide as a result of a frameshift mutation located approximately halfway through the coding sequence of the gene. This mutation is polar on expression of the downstream genes (ilvEDA) because transcription terminates within the translationally barren region that results from the mutation. Contrary to this, Salmonella typhimurium produces a full-length functional ilvG protein and is therefore unlikely to manifest this polarity event. E. coli K12 strains with mutations either in the ilvG gene (which restores a full-length protein) or in the rho gene, relieve this polarity suggesting that this event couples transcription and translation in a manner analogous to attenuation. This paper describes experiments designed to determine the molecular nature and location of the polarity event. Most significantly, this work establishes the contribution of the internal promoter (ilvEp, located downstream of the polar site) to the expression of the downstream genes in E. coli K12 wild-type and mutant strains (ilvG) and by extension to the role of this promoter in S. typhimurium. This analysis suggests that ilvEp contributes as much as 90% of ilvEDA expression in wild-type E. coli K12 and only 15% in wild-type S. typhimurium when grown under non-repressing conditions.

Point mutations in the regulatory region of the ilvGMEDA operon of Escherichia coli K-12.

The ilvGMEDA operon of Escherichia coli K-12 is preceded by a regulatory region containing a promoter, a leader, and an attenuator. This region has been extensively characterized biochemically. In this note point mutations of the regulatory region are reported. The effect of these mutations on expression from the ilv regulatory region supports the previous biochemical analysis.

Physical identification of an internal promoter, ilvAp, in the distal portion of the ilvGMEDA operon.

It has been previously demonstrated that the ilvGMEDA operon is expressed in vivo from the promoters ilvGp2 and ilvEp. An additional internal promoter is identified and designated ilvAp. This internal promoter, which allows independent expression of ilvA, has been analyzed both in vivo and in vitro. Our results indicate that: (1) ilvAp exists in both Escherichia coli K-12 and Salmonella typhimurium, as demonstrated by fusion to the galK reporter gene; (2) ilvAp is located in the distal coding sequence of ilvD; (3) the ilvAp sequences are not identical for these two bacterial species; (4) transcription from ilvAp of E. coli K-12 was demonstrated; (5) expression from ilvAp responds to the availability of oxygen; (6) potential 3' 5'-cyclic AMP receptor protein binding sites exist adjacent to ilvAp.

Binding of integration host factor (IHF) to the ilvGp1 promoter of the ilvGMEDA operon of Escherichia coli K12.

Crude extracts of Escherichia coli K-12 were found to bind DNA restriction fragments containing ilvGp1. Our analysis using a series of restriction fragments and a BamHI linker mutation indicate that a factor binds to ilvGp1 or adjacent to it. Analysis with mutant strains of E. coli K-12 and purified IHF indicate that IHF binds to ilvGp1. Furthermore, both analysis in vivo and in vitro indicate that IHF precludes transcription from ilvGp1.

Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase.

The nucleotide sequence of the gene, ilvA, for biosynthetic threonine deaminase (Tda) from Salmonella typhimurium was determined. The deduced amino acid sequence was compared with the deduced amino acid sequences of the biosynthetic Tda from Escherichia coli K-12 (ilvA) and Saccharomyces cerevisiae (ILV1) and the biodegradative Tda from E. coli K-12 (tdc). The comparison indicated the presence of two types of blocks of homologous amino acids. The first type of homology is in the N-terminal portion of all four isozymes of Tda and probably indicates amino acids involved in catalysis. The second type of homology is found in the C-terminal portion of the three biosynthetic isozymes and presumably is involved in either (i) the binding or interaction of the allosteric effector isoleucine with the enzyme, or (ii) subunit interactions. The sites of amino acid changes of two E. coli K-12 ilvA alleles with altered response to isoleucine are consistent with the conclusion that the C-terminal portion of biosynthetic Tda is involved in allosteric regulation.

The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12.

In this report we present the complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli. This operon contains five genes encoding four of the five enzymes required for the biosynthesis of isoleucine and valine. We identify and describe the coding regions for these five structural genes and the structural and functional features of the flanking and internal regulatory regions of this operon. This new information contributes to a more complete understanding of the overall control of the biosynthesis of isoleucine and valine.

Effect of the deletion of upstream DNA sequences on expression from the ilvGp2 promoter of the ilvGMEDA operon of Escherichia coli K-12.

Transcription in vitro of the regulatory region of the ilvGMEDA operon yields two attenuated RNAs initiated from the tandem promoters ilvGp1 and ilvGp2. Both S1 nuclease analysis and the fusion of ilvGp1 to galK indicate that transcription is not initiated in vivo from ilvGp1. However deletion of DNA sequences 150 to 100 bp upstream of ilvGp2 drastically reduces expression in vivo from ilvGp2. Both the distance separating ilvGp2 from the upstream DNA sequences and their relative orientation to each other on the DNA helix affect expression from ilvGp2. Deletion of DNA sequences approximately 400 bp upstream of ilvGp2 increases expression in vivo from this promoter. Analysis of products of transcription in vitro indicates that the effects observed in vivo are probably not due to DNA conformation or interactions of RNA polymerase.

Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons from Escherichia coli K-12 and Salmonella typhimurium.

It was previously determined that the distal portion of the ilvGMEDA operon was expressed despite the insertion of transposons into ilvG and ilvE. This observation suggested the existence of internal promoters upstream of ilvE (pE) and ilvD (pD). The internal promoter pE, responsible for part of ilvEDA expression, has been analyzed both in vivo and in vitro. Our results indicate that: pE exists in both E. coli K-12 and S. typhimurium; pE is located in the distal end of the ilvM coding sequence; the pE sequence is highly conserved in the two bacteria; the amino acid sequence of the ilvM gene product is 93% homologous between the two bacteria; transcription from pE can be demonstrated both in vivo and in vitro; the efficiency of pE is essentially equivalent in the two bacteria.

Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis.

A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined.

Restriction endonuclease analysis of the ilvGEDA operon of members of the family Enterobacteriaceae.

Four of the genes required for the biosynthesis of isoleucine and valine form the ilvGEDA operon in Escherichia coli K-12 and Salmonella typhimurium. The structural relationship of these genes was examined in eight other members of the family Enterobacteriaceae by genomic Southern blot hybridization. These genes are contiguous in all the strains examined, and specific restriction sites appear to be highly conserved, indicating the possible functional importance of the DNA sequences of which they are part.

Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12.

DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon. The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene. The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome. This is the first observation of this type of Tn5-mediated event. Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E. coli K-12 lacks enzyme activity. The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA. The detailed structure of these deletions should prove useful for the investigation of other genes in this region. This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.

Glucocorticoid inhibition of initiation of transcription of the DNA encoding rRNA (rDNA) in lymphosarcoma P1798 cells.

Cell-free extracts of lymphosarcoma P1798 cell culture lines support faithful initiation upon the cloned mouse DNA encoding rRNA (rDNA) promoter, whereas extracts from cells treated for 16 hr with 0.1 microM dexamethasone cannot. Extracts from both sources transcribe the cloned 5S RNA gene in vitro and mixing experiments further demonstrate that inhibition of transcription of rDNA in vitro is not due to nucleases or inhibitors of transcription present in extracts from glucocorticoid-treated cells. Incubation of extracts from control cells at 45 degrees C for 15 min inactivates RNA polymerase I and abolishes transcription. Activity can be restored by the addition of partially purified RNA polymerase I from control cells and hormone-treated cells. Moreover, extracts from hormone treated cells can be reconstituted by the addition of a partially purified, heat-stable transcription factor from control cells.

Characterization of a genomic clone for rat seminal vesicle secretory protein IV.

The entire coding region for rat seminal vesicle secretory protein IV was obtained on a 3.5 kb Eco RI fragment isolated from a genomic library in lambda Charon 4A. The coding sequence for SVS IV message is interrupted twice by introns. The first lies just downstream from the juncture of the 21 amino acid secretory signal peptide with the start of the mature protein, and the second lies in the 3'-nontranslated region. The major transcriptional start site was mapped by primer extention and is 22 nucleotides upstream from the translational initiation codon. S1 protection experiments indicated additional minor transcriptional starts about 27 and 50 nucleotides further upstream from the major cap site. The entire transcriptional unit comprises about 1740 nucleotides. The SVS IV gene does not belong to an obvious gene family, and it is conserved in mice and guinea pigs.

Isolation of recombinant bacteriophage containing male-specific mouse DNA.

The mammalian Y chromosome is an isolated piece of genetic material that directs sexual determination and gametogenesis. Very little is understood about the mechanism whereby the Y chromosome carries out these functions. Also, there is a severe lack of genetic markers on this chromosome. In order to understand the structure and function of the Y chromosome at the level of its DNA sequences and to provide genetic markers, we are isolating clones of DNA whose sequences are found primarily in DNA from male mice. To this end, we have developed a procedure for the identification of such clones. Application of this screening procedure to a lambda library derived from mouse sperm DNA has yielded 12 distinct clones, part of whose sequences are present predominantly in male DNA. Besides this DNA, they also contain other sequences that are shared with female DNA. These clones are either derived from the Y chromosome or they represent autosomal sequences specifically amplified during male development.

DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12.

Six ilvG (IlvG+) mutations of Escherichia coli K-12 were transferred to recombinant plasmids, and the DNA sequence of each mutation was determined. This analysis confirmed that expression of the ilvG gene product (acetohydroxy acid synthase II) requires the deletion of a single base pair or the addition of two base pairs within ilvG to displace a frameshift site present in wild-type E. coli K-12. This system should be useful in the analysis of potential frameshift mutagens.