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Background: Phosphoribosylaminoimidazole carboxylase (PAICS), a de novo purine metabolic enzyme, has been identified as an oncogene in several tumor types, including breast cancer and prostate cancer. However, the role of PAICS in human lung adenocarcinoma (LADC) requires further study., Methods: In this research, the effects of PAICS on the occurrence and prognosis of LADC were evaluated using integrative bioinformatics analyses., Results: By employing the bioinformatics analyses of several public databases, PAICS, which is overexpressed in the LADC tissues, was identified as a potential tumor-promoting gene in LADC biology. Several relevant clinical studies indicated that the upregulation of PAICS was statistically correlated with a shorter overall survival time. Moreover, the carcinogenic function of PAICS in LADC was validated by the further protein-protein interactions (PPI) and biological process annotation analysis. Mechanistically, we found that the PAICS methylation level was significantly lower in the LADC tissues compared to the normal lung tissues. Furthermore, using the MEXPRESS web tool, we predicted 15 possible DNA methylation sites in the nucleotide sequences of PAICS, which could explain the alteration in the PAICS expression levels in LADC., Conclusions: Our work demonstrates that high levels of PAICS are found in LADC and that this gene may be a potential therapeutic target for this subset of lung cancers. Determining the detailed roles of PAICS in LADC biology may provide useful information for further investigations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Arginine decarboxylase (ADC), one of the enzymes responsible for putrescine (Put) biosynthesis, has been shown to be implicated in stress response. In the current paper attempts were made to clone and characterize a gene encoding ADC from peach (Prunus persica (L.) Batsch, 'Akatsuki'). Rapid amplification of cDNA ends (RACE) gave rise to a full-length ADC cDNA (PpADC) with a complete open reading frame of 2178 bp, encoding a 725 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced PpADC protein sequence shared a high identity with ADCs from other plants, including several highly conservative motifs and amino acids. Southern blotting indicated that PpADC existed in peach genome as a single gene. Expression levels of PpADC in different tissues of peach (P. persica 'Akatsuki') were spatially and developmentally regulated. Treatment of peach shoots from 'Mochizuki' with exogenous 5 mM Put, an indirect product of ADC, remarkably induced accumulation of PpADC mRNA. Transcripts of PpADC in peach leaves from 'Mochizuki' were quickly induced, either transiently or continuously, in response to dehydration, high salinity (200 mM NaCl), low temperature (4 degrees C) and heavy metal (150 microM CdCl(2)), but repressed by high temperature 37 degrees C) during a 2-day treatment, which changed in an opposite direction when the stresses were otherwise removed with the exception of CdCl(2) treatment. In addition, steady-state of PpADC mRNA could be also transiently up-regulated by abscisic acid (ABA) in 'Mochizuki' leaves. All of these, taken together, suggest that PpADC is a stress-responsive gene and can be considered as a potential target that is genetically manipulated so as to create novel germplasms with enhanced stress tolerance in the future.

The cuticular melanization phenotype of black flies is rescued by beta-alanine, but beta-alanine production, by aspartate decarboxylation, was reported to be normal in assays of black mutants, and although black/Dgad2 is expressed in the lamina, the first optic ganglion, no electroretinogram (ERG) or other visual defect has been demonstrated in black flies. The purpose of this study was to investigate the black gene, and protein, in black(1) mutants of Drosophila melanogaster in order to resolve the apparent paradox of the black phenotype. Using black(1) mutant flies we show that (1) aspartate decarboxylase activity is significantly reduced in adults and at puparium formation, consistent with defects in cuticular and non-cuticular processes, (2) that the black(1) mutation is a frameshift, and black(1) flies are nulls for the black/DGAD2 protein, and (3) that behavioural experiments using Buridan's paradigm, demonstrate that black responds abnormally to visual cues. No ERG, or target recognition defects can be demonstrated suggesting a problem with higher order visual functions in black mutants.

Arginine decarboxylase (ADC) is a key enzyme involved in the synthesis of polyamines, which have been implicated in developmental processes and stress responses in higher plants. An ancestral ADC gene appears to have been duplicated at the origin of the Brassicaceae family, thus yielding two paralogues in the derived taxa. ADC gene structure was investigated in Pringlea antiscorbutica R. Br., a geographically isolated Brassicaceae species that is endemic from the subantarctic region. P. antiscorbutica exhibits several biochemical and physiological adaptations related to this cold and harsh environment, including high levels of polyamines, which is unusual in higher plants, and especially high levels of agmatine, the product of the ADC-catalysed reaction. Various ADC clones were obtained from P. antiscorbutica. Sequence and phylogenetic analysis showed that all these clones fitted with the presence of two paralogues, PaADC1 and PaADC2, in P. antiscorbutica. Expression of these ADC paralogues was analyzed in P. antiscorbutica during vegetative development and response to stress. Whereas PaADC2 was expressed at both seedling and mature stages, PaADC1 transcripts were hardly detected during early development and were significantly expressed in mature plants. Moreover, PaADC2, but not PaADC1, expression was up-regulated in response to chilling and salt stress at seedling stage. Analysis of 5' regulatory regions of these ADC genes revealed several differences in putative cis-regulatory elements, which could be associated with specific expression patterns. These results were compared to ADC paralogue expression in Arabidopsis thaliana and are discussed in the evolutionary context of genetic diversity resulting from gene duplication.

We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also constructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi.

A cluster of genes on megaplasmid pRmeSU47b, bhbA-D, is required for growth on the polyhydroxyalkanoate degradation pathway intermediates 3-hydroxybutyrate and acetoacetate as sole carbon source. DNA sequence analysis of the bhbA gene indicated that it encoded a protein of 712 amino acids (aa) (78kDa) which appeared to be a homodimeric methylmalonyl-CoA mutase enzyme (EC 5.4.99.2). Cell-free extract of a bhbA::Tn5 mutant was devoid of methylmalonyl-CoA mutase activity, thus confirming the identity of the bhbA-encoded enzyme. The reason for the requirement of methylmalonyl-CoA mutase activity for operation of the polyhydroxyalkanoate degradation pathway is not immediately apparent. Situated immediately upstream of bhbA, in the same orientation, is a gene which is predicted to encode a protein that exhibits remarkable sequence similarity to the alpha subunit of propionyl-CoA carboxylase (EC 6.4.1.3). A mutation in this gene did not affect ability to grow on 3-hydroxybutyrate as sole carbon source. Downstream of, and oriented towards bhbA, was identified a member of the GNTR class of transcriptional regulator-encoding genes. It is not yet known whether this regulatory protein is directly involved in modulation of bhbA expression.

YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerol kinase promoters. YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains. The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens. Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g. modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components). YIpDCE1 has been used to create strains simultaneously overexpressing the permease (FTR1) and oxidase (FET3) components of the yeast high-affinity iron uptake system. This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms.

The Candida glabrata ADE2 gene encoding aminoimidazole ribonucleotide (AIR) carboxylase (EC 4.1.1.21) was isolated by complementation of the ade2-1 mutation in Saccharomyces cerevisiae. The predicted amino acid (aa) sequence is 75% identical to that of S. cerevisiae. Integrative transformation was used to produce a C. glabrata strain bearing a deletion of ADE2 coding sequences. A high-copy-number shuttle vector bearing the ADE2 gene was constructed and contains a fragment of S. cerevisiae mitochondrial (mt) DNA that confers the ability to replicate autonomously in C. glabrata.

The degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde (CAA), a toxic intermediate in the cells if it is not metabolized further by the NAD(+)-dependent CAA dehydrogenases. Here, we describe the cloning, sequence and expression in Escherichia coli of aldA, a plasmid-located CAA dehydrogenase-encoding gene of GJ10 as well as a chromosomal homolog, designated aldB. The DNA-predicted amino acid (aa) sequences of the two proteins (505 aa in AldA and 506 aa in AldB) are 84% identical. The cloned aldA and aldB genes were verified by their expression in the E. coli T7 polymerase/promoter and the pUC lac promoter systems. The expression level of AldA and its enzymatic activity towards CAA were both higher than those of AldB. In a hybrid construct, the 3'end of aldB was able to complement, although not completely, the corresponding portion of aldA to produce a functional gene. Both AldA and AldB proteins of GJ10 share the highest degree of sequence identity with an acetaldehyde dehydrogenase (ALDH) encoded by acoD of Alcaligenes eutrophus (77.3-78% identity). Together with at least three other ALDHs of prokaryotic origin, these proteins apparently form a special class of ALDHs whose expressions are dependent on RpoN factors. By pulsed-field gel electrophoresis the 225-kb pXAU1 plasmid encoding aldA was shown to be linear.

Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs). The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E. coli. ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3.

A 2.91-kb fragment of the Escherichia coli chromosome containing the speC gene, encoding biosynthetic ornithine decarboxylase (ODC) was sequenced. The speC gene is encoded by a 2133-bp ORF; the deduced amino-acid sequence contains 711 residues whose predicted molecular mass is 79,505 Da.

The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids. Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation. The nucleotide sequence of the smallest complementing fragment was determined. The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli. PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype. PAD1 is a single-copy gene in the yeast genome and not essential for viability. The PAD1 locus was physically mapped to a position approx. 140 kb from the left end of chromosome IV.

The lysA and proC genes of Mycobacterium tuberculosis were cloned by screening of a recombinant lambda gt11 M. tuberculosis DNA library for phages able to complement lysA and proC Escherichia coli mutants. The lysA gene encodes diaminopimelic acid decarboxylase which catalyzes the conversion of diaminopimelic acid (DAP) to lysine. The lysA gene from M. tuberculosis encodes a 44-kDa protein, as determined by maxicell experiments. The nucleotide sequence of the structural gene was established. The deduced amino acid sequence was found to exhibit significant homology (from 55% to 73% similarity, and from 27% to 53% identity) to DAP decarboxylase sequences from other bacterial species.

The nucleotide sequence of three open reading frames in the acetone-production locus of Clostridium acetobutylicum ATCC824 has been established. The three gene products, corresponding to acetoacetate decarboxylase (EC 4.1.1.4) and both subunits of the acetoacetyl-CoA:acetate/butyrate:CoA transferase (EC 2.8.3.9) are transcribed in two convergently arranged operons. The intervening DNA region separating the two transcripts is characterized by an inverted repeat which appears capable of forming a stem-loop structure functioning as a Rho-independent transcription terminator in both directions.

Sequence analysis of a 2-kilobase DNA fragment from Rhodococcus sp. NI86/21 revealed an open reading frame encoding a 476-amino-acid protein with striking homology to the rat and human propionyl-CoA carboxylase beta subunits. The nucleotide sequence of a corresponding prokaryotic gene has not yet been reported. Upstream, the C-terminal part of a putative beta-ketoacyl synthase was identified.

Due to two different polyadenylation signals, two forms of S-adenosylmethionine decarboxylase (AdoMetDC) mRNA (2.1 and 3.4 kb) are present in human and rodent tissues. The nucleotide sequences of rat and human cDNAs corresponding to the shorter mRNA were published previously by us [Pajunen et al., J. Biol. Chem. 263 (1988) 17040-17049]. These sequences covered the coding regions but were incomplete at their 5' ends. Here we report the sequence of rat cDNA spanning the entire longer mRNA with a substantially extended leader region, and compare the sequence with that of a rat psi AdoMetDC pseudogene isolated from a rat genomic library. Relative to the mRNA, the pseudogene has multiple base changes as well as insertions, and deletions. Furthermore, it lacks introns, and is flanked by a short direct repeat. These are typical characteristics of a processed retrogene.

The ColE1 hybrid plasmid, pLC20-10, carrying the ppc gene and the argECBH gene cluster of Escherichia coli K-12, was characterized. The ppc gene coding for phosphoenolpyruvate carboxylase (EC 4.1.1.31), was subcloned into the plasmid pBR322 to give the plasmids pS2 and pS3. These plasmids carried a 4.4-kb SalI segment containing the ppc gene, in both orientations. The specific activity of the enzyme was increased approx. 20-fold by these plasmids. Experiments with maxicells harboring pS2 showed that the 90-kDal enzyme subunit was encoded by the plasmid. The location of the ppc gene in pS2 and the direction of transcription of the gene were determined. In DNA-DNA hybridization experiments using pS2 as a probe, significant hybridizations were observed with DNAs from E. coli strains K-12 and W, and from Salmonella typhimurium, but not with those from Chlorella regularis, Anacystis nidulans, Rhodospirillum rubrum, and Pseudomonas AM-1.

We have modified the transformation procedures of Ballance et al. [Biochem. Biophys. Res. Commun. 112 (1983) 284-289] to give increased rates of transformation in Aspergillus nidulans. With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5'-phosphate decarboxylase gene of A. nidulans, by transformation with a library of wild-type (wt) sequences in pBR329. We have recovered, by marker rescue from one such transformant, a plasmid (pJR15) that carries an A. nidulans sequence that complements pyrG89 efficiently. In three experiments, this plasmid gave an average of 1985 stable transformants/micrograms of transforming DNA. We have analyzed ten of these genetically and by Southern hybridization. In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant several copies of pJR15 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences. Sequence and S1 nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5'-phosphate decarboxylases of Neurospora crassa and Saccharomyces cerevisiae. We conclude that this gene is the wt pyrG allele. Finally, we have compared the 5'- and 3'-noncoding sequences and intron splice sequences to other genes of A. nidulans and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I.

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

The structural gene (pyrF) for orotidine-5'-phosphate decarboxylase (OMPase, EC 4.1.1.23) of Escherichia coli K-12 has been cloned as part of two PvuII fragments (1.2 and 0.9 kb) to form the recombinant plasmid pDK26. Extracts of E. coli [pDK26] had 80-fold higher levels of OMPase activity than wild-type strains without the plasmid. Maxicell analysis showed that pDK26 encoded two proteins of Mr 27 000 [pyrF(OMPase)] and 15 000 (Z) in addition to the ampicillin-resistance determinant. The approximate initiation site and direction of transcription of the pyrF gene have been determined. Extracts of strains that were deficient in polynucleotide phosphorylase (PNPase) had higher levels of OMPase activity than isogenic PNPase+ strains when one or two copies of the pyrF gene were present per cell either in the chromosome or on a low copy number plasmid. However, no significant difference in OMPase activity was seen in PNPase- strains that contained the pyrF gene cloned in a multicopy plasmid. Southern hybridization experiments showed that the yeast gene for OMPase (URA3) and the E. coli pyrF gene had less than 70% DNA sequence homology.

Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined. The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues. The codon usage of the ppc of A. nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A. nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E. coli ppc.

Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322. Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A. variabilis genomic library. A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A. variabilis PEPCase. E. coli HB101 cells transformed with plasmid pTRH1 have approx. 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A. variabilis PEPCase.

The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway. The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined. Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced. Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast. Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks. Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified.

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.

The gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase/Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced amino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon.

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.

The mouse dihydrofolate reductase gene and a segment of the Escherichia coli trp operon are expressed in Bacillus subtilis when cloned in the "expression plasmid" pPL608. The cloned mouse gene confers trimethoprim resistance on B. subtilis and the cloned trp fragment complements mutations in the B subtilis trpD, C and F genes Expression of both cloned fragments is dependent on a promoter present in the vector plasmid. The E. coli trp fragment is cloned in a HindIII site within a chloramphenicol acetyltransferase gene present on pPL608, and as a result, expression of the E. coli trpC gene product is inducible by chloramphenicol. The mouse gene is inserted at a PstI site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible. The replication functions and neomycin-resistance of pPL608 are derived from pUB110. Accordingly, pPL608 is stably maintained at high copy number in B. subtilis.

The expression of the cloned Saccharomyces cerevisiae URA3 gene in Escherichia coli on both plasmid and phage vectors was studied. Isolates of the gene from two different laboratory strains of yeast differ in their ability to be expressed in E. coli in the absence of external adjacent promoters of transcription. The DNA sequence of the two genes was determined and revealed several differences in the DNA flanking the structural gene. One base change alters the "Pribnow-box" of an E. coli promoter present in the yeast sequences. Three amber alleles of the yeast gene were also cloned from yeast. Two of the alleles could be suppressed in E. coli by a tRNA suppressor mutation. One of the amber alleles was determined to be a mutation in the seventh codon of the structural gene, thereby establishing the reading frame and extent of the coding sequence. The initiator codon of the reading frame encoding the URA3 structural gene is preceded by two other ATG codons in a different reading frame 61 and 79 bp away. The nearer ATG begins an open reading frame that overlaps the structural gene sequences by 17 bp. With the DNA sequence of the URA3 gene many of the common yeast vector plasmids are now completely known at the level of DNA sequence.

Genes involved in biosynthesis of the siderophore desferrioxamine B have been cloned from Streptomyces pilosus using a self-cloning approach with the low-copy plasmid pIJ943. Mutants in two steps of the pathway can be complemented by a 4.5-kb DNA fragment. One of these mutants is defective for the first step, lysine decarboxylation. Expression of lysine decarboxylase by the cloned DNA was found to be regulated by iron. S1 mapping and Northern blotting experiments show that iron regulation of desferrioxamine B production is exerted at the transcriptional level, and also that genes involved specify a long message, which determines at least two steps in the biosynthetic pathway.

We previously cloned the structural gene hdcA, which encodes the enzyme histidine decarboxylase (HDC; EC 4.1.1.22), from Lactobacillus 30a and found what appeared to be the start of a second gene 59 nucleotide (nt) downstream from the hdcA stop codon [Vanderslice et al., J. Biol. Chem. 32 (1986) 15186-15191]. Here we report the complete nt sequence of this second gene, which we have named hdcB, and show that it encodes a 20-kDa protein, HDCB, which was purified from Escherichia coli. The hdcA and hdcB genes together comprise an operon, the transcription from which is shown to be increased threefold by the presence of histidine in the growth medium. Western blots were used to quantitate the rise in concentrations of both gene products during histidine induction of the hdc operon. This increase was found to be proportional to the observed threefold increase in the concentration of the respective mRNAs. Transcription of the hdc operon in the mutant-3 strain of Lactobacillus 30a [Recsei and Snell, Biochemistry 12 (1973) 365-371] was shown to be constitutively 15-fold greater than in uninduced wild type cells and was unaffected by histidine. The transcription start point was defined as a guanine 73 nt 5' to the start codon of the hdcA gene. Of the transcripts initiated at this promoter, 15% include both hdcA and hdcB sequences, the remainder terminate in the intergenic region and thus encode only hdcA.

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.

The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library. The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation. The complete nucleotide sequence of the fragment containing the URA1 gene was determined. Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns. The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms. No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene.