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Nucleotide sequence of a gene, hpt, for hypoxanthine phosphoribosyltransferase from Vibrio harveyi
... encoding a potential 203-amino-acid polypeptide, designated HapR. BLAST searches using the DNA sequence and ORF translations identified a single homologue, the luxR gene and LuxR protein of V. harveyi (Showalter et al., 1990) (Fig. 2). Upstream of the hapR ORF was the 5Ј end of a divergently translated ORF (Fig. 1B) that showed homology to the hpt gene of V. harveyi, which is also upstream of and divergently oriented to the luxR gene in that organism (Showalter and Silverman, 1990). ...
... BLAST searches using the DNA sequence and ORF translations identified a single homologue, the luxR gene and LuxR protein of V. harveyi (Showalter et al., 1990) (Fig. 2). Upstream of the hapR ORF was the 5Ј end of a divergently translated ORF (Fig. 1B) that showed homology to the hpt gene of V. harveyi, which is also upstream of and divergently oriented to the luxR gene in that organism (Showalter and Silverman, 1990). ...
... Since the predicted polypeptides of HapR and LuxR are highly homologous and identical in the predicted DNAbinding domain, we tested to see if each gene could activate expression of the other's activatable promoter (i.e. the hap and lux operon promoters) in E. coli. A luxR clone (pRS156) and a functional but activator-requiring lux operon clone (pRS205) were obtained from Dr Michael Silverman (Showalter et al., 1990) and transformed into TX1[pQF3.1] and TX1[pC1.1], ...
The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Escherichia coli. We cloned and sequenced the 0.7kb hap promoter fragment from strain 3083-2 and showed that hap is located immediately 3' of ompW, encoding a minor outer membrane protein. A clone from a genomic library of strain 3083-2 was isolated, which was required for activation of the hap promoter in E. coli. Expression from the hap promoter only occurred late in the growth phase. A single complete open reading frame (ORF) designated HapR was identified on a 1.7 kb DNA fragment that was required for activation. Allelic replacements showed that hapR was also essential for hap expression in V. cholerae. In El Tor, but not in classical biotypes of V. cholerae, hapR mutations also produced a rugose colonial phenotype. HapR was shown to encode a 203-amino-acid polypeptide with 71% identity to LuxR of V. harveyi, an essential positive regulator of the lux operon that has no previously identified homologues. The amino-terminal domain (residues 21-68) showed significant homology to the TetR family of helix-turn-helix DNA-binding domains and was 95% identical to the same domain of LuxR. HapR and LuxR activated both the hap and the lux promoters at near wild-type levels, despite only limited homology in the promoter sequences (46% identity with 12 gaps over 420bp). DNA sequences and ORFs 5' (but not 3') of the hapR and luxR loci were homologous, suggesting a common origin for these loci, and hapR-hybridizing sequences were found in other vibrios. We conclude that HapR is absolutely required for hap expression and that HapR and LuxR form a new family of transcriptional activator proteins.
... Hfq has also been identified as a negative regulator of bioluminescence in V. harveyi (Lenz et al., 2004). Additional variants of interest were observed in gcvP, which plays a role in the virulence of V. cholerae in Drosophila hosts (Vanhove et al., 2017), and in hpt, which is located just upstream of the quorum-sensing regulating gene luxR in V. harveyi (Showalter and Silverman, 1990). The bacterial genes involved in the S. tubifer-P. ...
All organisms depend on symbiotic associations with bacteria for their success, yet how these interspecific interactions influence the population structure, ecology, and evolution of microbial symbionts is not well understood. Additionally, patterns of genetic variation in interacting species can reveal ecological traits that are important to gene flow and co-evolution. In this study, we define patterns of spatial and temporal genetic variation of a coral reef fish, Siphamia tubifer, and its luminous bacterial symbiont, Photobacterium mandapamensis in the Okinawa Islands, Japan. Using restriction site-associated sequencing (RAD-Seq) methods, we show that populations of the facultative light organ symbiont of S. tubifer exhibit genetic structure at fine spatial scales of tens of kilometers despite the absence of physical barriers to dispersal and in contrast to populations of the host fish. These results suggest that the host’s behavioral ecology and environmental interactions between host and symbiont help to structure symbiont populations in the region, consequently fostering the specificity of the association between host generations. Our approach also revealed several symbiont genes that were divergent between host populations, including hfq and a homolog of varS, both of which play a role in host association in Vibrio cholerae. Overall, this study highlights the important role that a host animal can play in structuring the distribution of its bacterial symbiont, particularly in highly connected marine environments, thereby promoting specificity of the symbiosis between host generations.
... Hypoxanthine phosphoribosyltransferase [HPRT; IMP: pyrophosp hate phosphoribosyltransferase, ES 2.4.2.8, also hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT)] is. a purine salvage enzyme that has been studied extensively. Complementary DNA (cDNA) encoding the human, schistosomal, malarial, trypanosomal, tritrichomonal, and bacte rial HPRT's have been cloned and sequenced ( [27][28][29][30][31][32], respectively). The human HPRT has been a subject of extensive investigation because defects in this enzyme are known to be responsible for genetically inhe rited gout and Lesch-Nyhan syndrome in humans [33,34]. ...
Giardia lamblia is one of the most ancient eukaryotes identified to date. It lacks de novo purine biosynthesis and is thought to rely solely on the functions of two salvage enzymes, adenine and guanine phosphoribosyltransferases (APRTase and GPRTase). We have cloned the gene encoding the G. lamblia GPRTase by complementation of the E. coli strain SØ609 (Δgpt-pro-lac, thi, hpt, pup, purH,J, strA) with a genomic library consisting of Sau3AI-digested G. lamblia DNA inserted into the Bluescript™ vector. Transformed SØ609 colonies grew on minimal medium supplemented with guanine at a frequency of 3.3 × 10−5 ampicillin-resistant colonies, but were unable to salvage hypoxanthine or xanthine, as predicted from previous studies of the native G. lamblia GPRTase. The sequence analysis of cloned DNA fragments reveals an open reading frame of 690 bp, encoding a protein of 26.3 kDa with an estimated pI of 6.83, in agreement with the reported subunit molecular weight of the native G. lamblia GPRTase. The deduced protein has less than 20% sequence identity to the human and other known HGPRTases, and features several significant changes in the primary sequence of the putative active sites of the enzyme, which may reflect the stringent substrate specificity of GPRTase. The recombinant GPRTase was expressed in E. coli and purified to >95% homogeneity. Kinetic studies of the recombinant enzyme showed an apparent Km of 74 μM for guanine. Hypoxanthine as an alternate purine substrate was used only when present in millimolar amounts, and xanthine was not utilized at all. This Giardia enzyme is thus a highly unique purine PRTase without a known parallel in any other living organisms.
Early studies involving purine salvage in Salmonella typhimurium resulted in the isolation and identification of a mutant strain possessing a genetically modified hypoxanthine phosphoribosyl-transferase (HPRT) with enhanced substrate specificity for guanine [Benson, C. E., and Gots, J. S. (1975) J. Bacteriol. 121, 77-82]. To explore the molecular basis for this altered substrate specificity in the mutant hpt gene product, degenerate oligonucleotide primers, designed according to the N- and C-termini of the HPRT of Escherichia coli, were used in polymerase chain reactions to amplify both the mutant and wild-type S. typhimurium hpt genes from genomic DNA. Analysis of the deduced amino acid sequences revealed that a single base mutation resulted in the encoding of a Thr in the mutant HPRT, instead of an Ile found in the wild-type enzyme, at a position analogous to position 192 (Leu-192) of the human HPRT. Comparison of kinetic data for purified recombinant mutant and wild-type HPRTs showed no difference in the overall catalytic efficiency (kcat/K(m)) with hypoxanthine as substrate, but with guanine, the mutant enzyme exhibited a more than 50-fold higher kcat/K(m) largely as a result of a decrease of nearly 2 orders of magnitude in K(m). Involvement in substrate binding of the cognate amino acid at position 192 in the human HPRT was investigated using site-directed mutagenesis. Mutation of Leu-192 to Thr did not significantly alter kcat/K(m) values for hypoxanthine and guanine compared to wild-type, and replacement of Leu-192 with Ile had no significant change in kinetics for either hypoxanthine or PRPP. However, this Ile substitution resulted in an over 15-fold decrease in the kcat/K(m) for guanine due to a greater than 15-fold increase in K(m). These results demonstrate that a single active site amino acid substitution in HPRTs can significantly alter the specificity for binding guanine.
Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.
Five purine auxotrophic mutants of Lactococcus lactis were isolated. L. lactis was capable of converting adenine, guanine and hypoxanthine to AMP, GMP and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activities. A 1.3 kb DNA fragment from L. lactis was cloned by complementation of the hpt mutation in Escherichia coli. Introduction of this fragment into L. lactis resulted in an increase in HGPRT activity. In vitro transcription and translation analysis showed that the fragment coded for a polypeptide with M(r) of 22,000. The nucleotide sequence of this hpt gene was determined.
We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed.
The deduced 182 amino acid sequence of an open reading frame in the photosynthetic bacterium Rhodobacter capsulatus shows significant similarity to the hypoxanthine-guanine phosphoribosyltransferases of other organisms. This similarity includes conserved amino acid residues involved in Lesch-Nyhan syndrome.
The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme in Trypanosoma cruzi is a rational target for the treatment of Chagas disease. To evaluate the T. cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T. cruzi DNA and sequenced. Translation of the nucleotide sequence of the hgprt revealed an open reading frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino acids. The T. cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid sequence identity to its human, Plasmodium falciparum, and Schistosoma mansoni counterparts, respectively, but was 50% identical to the T. brucei HGPRT protein. Northern analysis of T. cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that hgprt was a single copy gene within the T. cruzi genome. The T. cruzi hgprt was inserted into the pBAce expression plasmid and transformed into Escherichia coli that are deficient in hypoxanthine and guanine phosphoribosylating activities. High levels of soluble, enzymatically active T. cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies. The recombinant HGPRT was purified to apparent homogeneity by GTP-agarose affinity chromatography and recognized hypoxanthine, guanine, and allopurinol, but not adenine or xanthine, as substrates. The availability of the hgprt clone and large amounts of pure HGPRT protein provide a foundation for a structure-based drug design strategy for the treatment of Chagas disease.
We have sequenced and studied the expressed protein of an HPRT mutation characterized by 5-12% residual erythrocyte activity, for which affected males exhibit hyperuricemia, arthritis and renal disease but are without severe neurological involvement. The HPRTMoose Jaw mutation is due to a single C to G transversion at nucleotide 582 relative to initiation of translation corresponding to substitution of aspartate 194 by glutamate. The mutant and wild type proteins were expressed and purified using the bacterial expression vector, pMAL-c2. The Km for hypoxanthine was increased 12-fold from 0.94 +/- 0.26 to 11.5 +/- 1.3 microM for control and mutant respectively. The apparent Km for PP-ribose-P was increased 44-fold from 6.8 +/- 0.6 to 295 +/- 7 microM for control and mutant respectively. Although the kcat of the mutant protein was equivalent to wild type, the catalytic efficiency, kcat/Km, of the purified mutant protein was only 6 and 3% of wild type with hypoxanthine and PP-ribose-P respectively. The mutant protein also exhibited positive cooperativity with PP-ribose-P, having a Hill coefficient of 2.3. The decreased substrate affinities and PP-ribose-P associated cooperativity of HPRTMoose Jaw provide additional evidence for the influence of carboxy-terminal residues of HPRT in specific catalytic functions.
We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is essential in nucleic acid metabolism of T. foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T. foetus. Northern blot analysis identified a single mRNA band of approximately 700-800 bases. The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid sequence with an estimated size of 21.1 kDa. The sequence is only 27.3% identical to that of the human HGPRTase. The T. foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E. coli. This construct yields completely soluble and enzymatically active recombinant T. foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E. coli. It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame. The high expression of this apparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy. To our knowledge, this is also the first time a gene from T. foetus was cloned and expressed.
A hypoxanthine-guanine phosphoribosyltransferase-encoding gene (HGPRT) from Toxoplasma gondii has been isolated and sequenced. The identity of the enzyme was determined by sequence comparison and complementation in a mutant Escherichia coli.
The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic
diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein
of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21 - 23 amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous
to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed
to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active
recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant
enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by
a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulate molecular and biochemical
system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases
of parasitic origin.
Analysis of Vibrio harveyi dark autoinducer mutants has demonstrated that the level of LuxR was much lower than that observed in wild-type cells. Complementation with luxR fully restored luminescence suggesting that the lux autoinducer may control expression of the luxR regulatory gene. By primer extension, the transcriptional start site of luxR was located 78 bp from the initiation codon. The level of the primer-extended product was enhanced upon addition of the lux autoinducer to the autoinducer mutants, which was confirmed by hybridization of lux mRNA with specific probes. By using chloramphenicol acetyltransferase as a reporter gene in a transcriptional fusion with luxR, the stimulatory effect of autoinducer on luxR expression was shown to occur at the level of the luxR promoter. The results provide evidence that the autoinducer signal in V. harveyi can be transduced through luxR, resulting in stimulation of luminescence.
We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRT protein. Transfection of HPRT-deficient mouse LA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.
Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was contransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.
Resistance to purine analogues in mutants isolated from Salmonella typhimurium, strain LT-2, resulted in the loss of specific purine nucleotide pyrophosphorylases. Thus, mutants resistant to 2,6-diaminopurine, 6-mercaptopurine, and 8-azaguanine lacked adenylic, inosinic and guanylic pyrophosphorylases, respectively. The mutants resistant to 2,6-diaminopurine were of two types. One of them, dap-r-3, was deficient in adenylic pyrophosphorylase, whereas in the other mutant, dap-r-6, activity of this enzyme was unaffected. However, both the mutants were unable to make corresponding nucleotides from 2,6-diaminopurine. Resistance to 6-mercaptopurine also resulted in two types of mutations. One type, mp-r-I, was deficient only in inosinic pyrophosphorylase; the other, mp-r-2, in both inosinic and guanylic pyrophosphorylases. Resistance to 8-azaguanine in the mutant, azg-r, resulted in loss of only guanylic pyrophosphorylase activity. All the mutants retained xanthylic pyrophosphorylase activity. Results indicated the existence of at least three (possible four) purine nucleotide pyrophosphorylases. Resistance to all the purine analogues was characterized by the inability of the resistant strains to make nucleotides from the analogues. These observations suggested that the nucleotides were probably the active inhibitory forms of the analogues, and that a deficiency in activating enzymes led to the resistance in the above mutants.
Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.
Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
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