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PubMed1997

Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.

Adenosine Triphosphatases↗
PubMed2001

Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

Acyltransferases↗
PubMed2003

Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.

Amino Acid Sequence↗
PubMed2005

Evolution of transcription regulatory genes is linked to niche specialization in the bacterial pathogen Streptococcus pyogenes.

Streptococcus pyogenes is a highly prevalent bacterial pathogen, most often giving rise to superficial infections at the throat or skin of its human host. Three genotype-defined subpopulations of strains exhibiting strong tropisms for either the throat or skin (specialists) or having no obvious tissue site preference (generalists) are recognized. Since the microenvironments at the throat and skin are distinct, the signal transduction pathways leading to the control of gene expression may also differ for throat versus skin strains of S. pyogenes. Two loci (mga and rofA/nra) encoding global regulators of virulence gene expression are positioned 300 kb apart on the genome; each contains alleles forming two major sequence clusters of approximately 25 to 30% divergence that are under balancing selection. Strong linkage disequilibrium is observed between sequence clusters of the transcription regulatory loci and the subpopulations of throat and skin specialists, against a background of high recombination rates among housekeeping genes. A taxonomically distinct commensal species (Streptococcus dysgalactiae subspecies equisimilus) shares highly homologous rof alleles. The findings provide strong support for a mechanism underlying niche specialization that involves orthologous replacement of regulatory genes following interspecies horizontal transfer, although the directionality of gene exchange remains unknown.

Bacterial Proteins↗
PubMed2006

Role for the Ssu72 C-terminal domain phosphatase in RNA polymerase II transcription elongation.

The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

Alanine↗
PubMed2005

Pharmacotherapy and pharmacogenetics of nicotine dependence.

The authors review recent advances in the pharmacotherapy and pharmacogenetics of nicotine dependence. Despite the negative health consequences of smoking, approximately 23% of adults in the United States are daily tobacco smokers and approximately 13% are nicotine dependent. Data for development of new medications for nicotine dependence are likely to come from animal models of the reinforcing value of nicotine, studies to identify proteins in transgenic rodents, and pharmacological studies of nicotine withdrawal. The initial pharmacogenetic studies of pharmacotherapies approved by the United States Food and Drug Administration for treatment of nicotine dependence-nicotine replacement (nicotine gum, nicotine nasal spray, and transdermal nicotine) and bupropion-have identified candidate alleles at the dopamine D2 receptor gene and mu opioid receptor gene that may predict therapeutic response. Because no one medication is likely to be safe and efficacious for a majority of persons with nicotine dependence, it will be useful to develop genetics-based methods and other tools to predict therapeutic response in subgroups of nicotine-dependent persons.

Administration, Cutaneous↗
PubMed2005

Increasing Fgf4 expression in the mouse limb bud causes polysyndactyly and rescues the skeletal defects that result from loss of Fgf8 function.

A major function of the limb bud apical ectodermal ridge (AER) is to produce fibroblast growth factors (FGFs) that signal to the underlying mesenchyme. Previous studies have suggested that of the four FGF genes specifically expressed in the mouse AER, Fgf8 is unique not only in its expression pattern, but also because it is the only such FGF gene that causes limb skeletal abnormalities when individually inactivated. However, when both Fgf8 and Fgf4 are simultaneously inactivated in the AER, the limb does not develop. One possible explanation for these observations is that although both of these FGF family members contribute to limb development, Fgf8 has functions that Fgf4 cannot perform. To test this hypothesis, we used a novel method to substitute Fgf4 for Fgf8 expression in the developing limb bud by concomitantly activating a conditional Fgf4 gain-of-function allele and inactivating an Fgf8 loss-of-function allele in the same cells via Cre-mediated recombination. Our data show that when Fgf4 is expressed in place of Fgf8, all of the skeletal defects caused by inactivation of Fgf8 are rescued, conclusively demonstrating that FGF4 can functionally replace FGF8 in limb skeletal development. We also show that the increase in FGF signaling that occurs when the Fgf4 gain-of-function allele is activated in a wild-type limb bud causes formation of a supernumerary posterior digit (postaxial polydactyly), as well as cutaneous syndactyly between all the digits. These data underscore the importance of controlling the level of FGF gene expression for normal limb development.

Animals↗
PubMed2006

Uncoupling dorsal-mediated activation from dorsal-mediated repression in the Drosophila embryo.

The Rel family transcription factor Dorsal patterns the dorsoventral axis of the Drosophila embryo by activating genes such as twist and snail and repressing genes such as decapentaplegic and zerknüllt. Dorsal represses transcription by recruiting the co-repressor Groucho. However, repression occurs only when Dorsal-binding sites are close to binding sites for other factors that also bind Groucho. The need for additional factors to assist Dorsal in repression may result from the intrinsically weak interaction between Dorsal and Groucho. To test this idea, we generated a Dorsal variant containing a high-affinity Groucho recruitment motif at its C terminus. As predicted, this variant functions as a dedicated repressor, silencing decapentaplegic and zerknüllt while failing to activate twist and snail. We also converted Dorsal into a dedicated activator by replacing its weak Groucho-recruitment motif with heterologous activation domains. Although the dedicated activator alleles fail to repress decapentaplegic and zerknüllt in the syncytial blastoderm embryo, they are able to pattern the dorsoventral axis. This indicates that dorsoventral patterning is not dependent upon Dorsal-mediated repression, reflecting the existence of redundant mechanisms to block Decapentaplegic signaling.

Animals↗
PubMed2006

Parasexual genetics using axenic cells.

Normally, vegetative Dictyostelium grow as haploid cells. Occasionally, two haploid cells fuse together during normal growth, forming a diploid cell containing both parental sets of chromosomes within a single nucleus. The diploid state is reasonably stable, and the growth, development, and general behavior of diploids are similar to their haploid parents. However, during normal growth of diploids, cells may spontaneously lose one copy of each chromosome at random and revert back to a haploid state containing a selection of chromosomes from both parents. This diploid cycle therefore allows nonsexual recombination between two different mutant strains. Diploid cells have multiple practical uses. They allow the generation of double and multiple knockouts, and are particularly useful for strains that are sick or difficult to generate using molecular genetics. They provide a means of manipulating genes that are lethal when disrupted in haploids. In diploids, it is possible to isolate heterozygous knockouts with no phenotype and then introduce a further mutant allele. These cells can then be segregated to yield haploid progeny with an effective gene replacement. Similarly, diploids made from different parent strains offer a means of examining the effects of different genetic backgrounds and overriding strain-specific phenotypes. A number of other uses are possible, making parasexual genetics potentially even more versatile.

Animals↗
PubMed1999

Association of neonatal hyperbilirubinemia with bilirubin UDP-glucuronosyltransferase polymorphism.

OBJECTIVE: The incidence of nonphysiologic neonatal hyperbilirubinemia is twice as high in East Asians as in whites. We studied whether the condition was associated with mutations in the gene for bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1), a key enzyme of bilirubin catabolism. DESIGN: We analyzed the UGT1A1 gene in 25 Japanese neonates who had nonphysiologic hyperbilirubinemia (serum bilirubin >257 micromol/L) with no obvious cause. They had all received phototherapy. The background control population consisted of 50 Japanese neonates whose transcutaneous jaundice index was monitored during the first week of life. We detected mutations by direct sequencing of polymerase chain reaction-amplified fragments of the gene. RESULTS: We found a polymorphism for UGT1A1 in exon 1; a G-->A transition at nucleotide 211 caused arginine to replace glycine at position 71 of corresponding protein product (G71R). The frequency of the mutated allele in the hyperbilirubinemic group (0.34) was significantly higher (chi2 = 5.56) than in the control group (0.16). In the control group the peak transcutaneous jaundice index of the carriers of G71R was significantly higher than it was in the normal infants. CONCLUSIONS: The missense mutation causing G71R is the first reported polymorphism for UGT1A1, and the mutation is a risk factor for nonphysiologic neonatal hyperbilirubinemia. The high incidence of hyperbilirubinemia in the Japanese may be attributable to the high frequency of this missense mutation.

Alleles↗
PubMed1983

Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase.

Growth of a temperature-sensitive general fatty acid synthesis mutant of Escherichia coli K12 at its restrictive temperature in the presence of exogenous palmitate results in lysis of the bacterium. Under these conditions, palmitate is incorporated into membrane phospholipid to a high level. Mutants of bacteria restricting this incorporation (having a palmitate-resistant phenotype) have been isolated and one such mutant, strain L8-2/3, has been further characterized. This mutant has lowered acyl-CoA synthetase (fadD) activity (25-33% of normal) and consequently is defective in fatty acid uptake. This lowered uptake could explain the palmitate-resistant phenotype of strain L8-2/3. However, both in vivo (fatty acid composition and positional distribution data) and in vitro (acyltransferase activity measurements) experiments suggest that this mutant is also altered in its acyltransferase activities. The mutation(s) of strain L8-2/3 appears to allow increased (approximately 2-fold) incorporation of myristate (and possible unsaturated fatty acids) into position 2 of 1-acyl-sn-glycerol 3-phosphate but normal palmitate incorporation into the same position. The incorporation of palmitate, myristate, and oleate into position 1 of sn-glycerol 3-phosphate by strain L8-2/3 is also higher than that observed with the parent, strain L8-2. Replacing the partially defective fadD gene of strain L8-2/3 with a wild type allele conferred on this strain the palmitate sensitivity and the acyltransferase activity of the parent strain L8-2. This finding, taken together with other data, suggests that acyl-CoA synthetase interacts with the acyltransferase(s) in some manner to influence the fatty acid specificity of the acyltransferase.

Acyltransferases↗
PubMed1995

Identification of guanine exchange factor key residues involved in exchange activity and Ras interaction.

We have carried out a functional analysis of the human HGRF55 exchange factor in the yeast Saccharomyces cerevisiae. Twelve residues conserved among most of all known guanine exchange factors (GEFs) have been independently changed to alanine. Taking advantage of the ability of Hgrf55p to replace the yeast Cdc25p exchange factor, and using the two-hybrid system with RAS2ala22 allele, we have identified key residues for the interaction with Ras and/or its activation. Substitution of arginine 392 to alanine leads to a complete loss of interaction with Ras, though the protein remains stable. Substitution of Asp266 or Arg359 to alanine results in inactive proteins at 39 degrees C, still able however to interact with Ras. The other charged-to-alanine substitutions led to no detectable phenotype when present alone but most of them dramatically increased the temperature sensitive phenotype observed with [Asp266Ala] substitution. Surprisingly, the cysteine to alanine substitution in the highly conserved PCVPF/Y motif proved to be without effect, suggesting that the sulfhydryl group is not essential for stability or interaction with Ras.

Alanine↗
PubMed2001

Polymorphic short tandem repeats for diagnosis of the Charcot-Marie-Tooth 1A duplication.

BACKGROUND: A 1.5-Mb microduplication containing the gene for peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 is responsible for 75% of cases of the demyelinating form of Charcot-Marie-Tooth disease (CMT1A). Methods for molecular diagnosis of CMT1A use Southern blot and/or amplification by PCR of polymorphic poly(AC) repeats (microsatellites) located within the duplicated region, or the detection of junction fragments specific for the duplication. Difficulties with both strategies have led us to develop a new diagnostic strategy with highly polymorphic short tandem repeats (STRs) located inside the CMT1A duplicated region. METHODS: We tested 10 STRs located within the duplication for polymorphic behavior. Three STRs were selected and used to test a set of 130 unrelated CMT1A patients and were compared with nonduplicated controls. The study was then extended to a larger population of patients. Alleles of interest were sequenced. A manual protocol using polyacrylamide electrophoresis and silver staining and an automated capillary electrophoresis protocol to separate fluorescently labeled alleles were validated. RESULTS: We identified three new STRs covering 0.55 Mb in the center of the CMT1A duplication. One marker, 4A, is located inside the PMP22 gene. The two others, 9A and 9B, more telomerically positioned, have the highest observed heterozygosity reported to date for CMT1A markers: 0.80 for 9A, and 0.79 for 9B. Tetra- and pentanucleotide repeats offered clear amplification, accurate sizing, and easy quantification of intensities. CONCLUSIONS: Combined use of the three STRs allows robust diagnosis with almost complete informativeness. In our routine diagnosis for CMT1A, they have replaced the use of other polymorphic markers, either in a manual adaptation or combined with fluorescence labeling and allele sizing on a DNA sequencer.

Charcot-Marie-Tooth Disease↗
PubMed2001

Initiation of enzyme replacement therapy for an adult patient with asymptomatic type 1 Gaucher's disease.

A 27-year-old woman was admitted for further examination of thrombocytopenia. Symptoms were absent, but physical examination demonstrated hepatosplenomegaly without neurological abnormalities. Bone marrow examination revealed many Gaucher cells, and glucocerebrosidase activity from cultured skin fibroblasts was markedly reduced. A 1448C (L444P) mutation was detected on one allele of the glucocerebrosidase gene. Because magnetic resonance imaging (MRI) of the femora indicated severe infiltration of Gaucher cells into bone marrow, enzyme replacement therapy was initiated despite the absence of skeletal symptoms. Hematologic abnormalities, visceral and bone involvement have been improving. In cases of thrombocytopenia or hepatosplenomegaly, Gaucher's disease should be suspected.

Adult↗
PubMed1997

Two mutations in the same low-density lipoprotein receptor allele act in synergy to reduce receptor function in heterozygous familial hypercholesterolemia.

Mutations in genes are not necessarily pathogenic. Expression of mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low-density lipoprotein (LDL) receptor gene: a missense Asn543. His mutation (N543H) in exon 11, and an in-frame 9-bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected.

Alleles↗
PubMed1998

Random amplification of polymorphic DNA and microsatellite genotyping of pre- and posttreatment isolates of Candida spp. from human immunodeficiency virus-infected patients on different fluconazole regimens.

Twelve patients infected with the human immunodeficiency virus (HIV) and with CD4 cell counts below 100 cells/microliter received fluconazole daily (200 mg; five patients) or weekly (400 mg; seven patients) for fungal prophylaxis during a 6-month period. Oropharyngeal swabs were taken at regular intervals in order to detect colonization with Candida spp. All yeast isolates were examined with respect to the development over time of fluconazole resistance. Genetic diversity among the strains was assessed in order to discriminate between selection of a resistant subclone and patient recolonization. Genotyping was performed through random amplification of polymorphic DNA (RAPD) analysis. Specific site polymorphisms were assayed by tracking length variability in several microsatellite loci. Finally, to maximize resolution, one of these loci (ERK1) was analyzed by nucleotide sequencing. Although the number of strains analyzed was too small to allow statistical verification, it appeared that when fluconazole was given weekly, a smaller fraction of the strains showed diminished sensitivity than when it was given daily. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain was seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor differences were seen in either the RAPD genotype or the microsatellite allele composition during the course of treatment. In general, microsatellite sequence data is in agreement with data obtained by other methods, but occasionally within-patient heterogeneity is indicated. The present results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or undergo small changes. Within a patient there may be different levels of intrastrain variation.

AIDS-Related Opportunistic Infections↗
PubMed2002

HLA-E allelic variants. Correlating differential expression, peptide affinities, crystal structures, and thermal stabilities.

Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (E(R)) is replaced by a glycine in HLA-E*0103 (E(G)). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-E(G) complexed with two distinct peptides were determined, and both were compared with the HLA-E(R) structure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.

Alleles↗
PubMed1990

Two new sheep haemoglobins, one of which is replaced by haemoglobin C in anaemia.

Two new haemoglobin variants, provisionally named Hb G and Hb H, were found during a survey of 295 Welsh Mountain cross-bred sheep. Both haemoglobins appear to be beta chain variants controlled by genes allelic to those for the common forms Hb A and Hb B. Studies on an anaemic Hb AH and an Hb AG type sheep showed that Hb G, like Hb A, is replaced by Hb C in anaemia whereas Hb H, like Hb B, is not replaced.

Anemia↗