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At least 379 records · Page 21Linked to original sources
PubMed1997

A genetic and functional analysis of the unusually large variable region in the M.AluI DNA-(cytosine C5)-methyltransferase.

The M.AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5'-AGCT-3'. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more "target-recognizing domains" (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M.AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M.AluI is in fact monospecific it was found that AGCT methylation represents only 80-90% of the methylating activity of this enzyme, while control experiments with the enzyme M.HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M.AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M.AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M.AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.

Amino Acid Sequence↗
PubMed2000

Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium.

The two-component regulatory system PmrA-PmrB confers resistance of Salmonella spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), bactericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed pmrE and pmrHFIJKLM. Both pmrE and pmrHFIJKLM produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more positively charged lipopolysaccharide (LPS) and thus reduces cationic AP binding. Experiments were conducted to further analyze the regulation of the pmrHFIJKLM operon and the role of this operon and the surrounding genomic region in LPS modification and antimicrobial peptide resistance. The pmrHFIJKLM genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The pmrHFIJKLM promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated pmrCAB promoter. Construction of nonpolar mutations in the pmrHFIJKLM genes showed that all except pmrM were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (pmrG and pmrD) were not necessary for PM resistance, but pmrD was shown to be regulated by the PhoP-PhoQ regulatory system. BALB/c mice inoculated with pmrA and pmrHFIJKLM mutant strains demonstrated virulence attenuation when the strains were administered orally but not when they were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tissues.

Animals↗
PubMed2002

Immature CD34+CD19- progenitor/stem cells in TEL/AML1-positive acute lymphoblastic leukemia are genetically and functionally normal.

One important question in stem cell biology of childhood acute lymphoblastic leukemia (ALL) is whether immature CD34+CD19- cells are part of the leukemic cell clone. CD34+CD19- cells from the bone marrow of 9 children with TEL/AML1-positive ALL were purified by flow sorting and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization, and methylcellulose cultures. In 3 of 8 patients analyzed by RT-PCR, no TEL/AML1-positive cells could be detected in the CD34+CD19- cell fraction. Altogether, the percentage of TEL/AML1-positive cells was low: 1.6% (n = 8; SD 2.2%) by nested real-time RT-PCR and 2.5% (n = 5; SD 2.6%) by fluorescence in situ hybridization. This correlated with the percentage of contaminating CD19+ leukemic cells in the CD34+CD19- cell fraction in 6 control sorts (mean 4.6%, SD 3.6%), indicating that the low levels of leukemic cells detected in the CD34+CD19- cell fraction could be attributed to sorter errors. Methylcellulose cultures in 3 patients provided further evidence that CD34+CD19- cells represent a candidate normal cell population. The clonogenicity of the CD34+CD19- cell fraction was similar to normal progenitors, including growth of primitive granulocyte, erythroid, macrophage, megakaryocyte colony-forming units. Each of 92 colonies from cultures with CD34+CD19- cells tested negative for TEL/AML1. In conclusion, our data support the hypothesis that the leukemia in TEL/AML1-positive childhood ALL originates in a CD19+ lymphoid progenitor. This has many therapeutic implications, eg, for purging of autologous stem cell products, flow cytometric monitoring of minimal residual disease, and targeting immunotherapy against the leukemic cell clone.

Antigens, CD19↗
PubMed2024

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation.

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Humans↗
PubMed1997

Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.

Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.

Animals↗
PubMed2006

The LRRK2 Gly2385Arg variant is associated with Parkinson's disease: genetic and functional evidence.

Evidence of LRRK2 haplotypes associated with Parkinson's disease (PD) risk was recently found in the Chinese population from Singapore, and a common LRRK2 missense variant, Gly2385Arg, was independently detected as a putative risk factor for PD in the Chinese population from Taiwan. To test the association between the Gly2385Arg variant in a large case-control sample of Chinese ethnicity from Singapore, and to perform functional studies of the wild type and Gly2385Arg LRRK2 protein in human cell lines. In a case-control study involving 989 Chinese subjects, the frequency of the heterozygous Gly2385Arg genotype was higher in PD compared to controls (7.3 vs. 3.6%, odds ratio = 2.1, 95% CI: 1.1-3.9, P = 0.014); these values yield an estimated population attributable risk (PAR) of approximately 4%. In a multivariate logistic regression analysis with the disease group (PD vs. controls) as the dependent variable and the genotype as an independent factor with adjustments made for the effect of age and gender, the heterozygous Gly2385Arg genotype remained associated with an increased risk of PD compared to wild type genotype (odds ratio = 2.67, 95% CI: 1.43-4.99, P = 0.002). The glycine at position 2385 is a candidate site for N-myristoylation, and the Gly2385Arg variant replaces the hydrophobic glycine with the hydrophilic arginine, and increases the net positive charge of the LRRK2 WD40 domain. In transfection studies, we demonstrated that both the wild type and Gly2385Arg variant LRRK2 protein localize to the cytoplasm and form aggregates. However, under condition of oxidative stress, the Gly2385Arg variant was more toxic and associated with a higher rate of apoptosis. Our study lends support to the contention that the Gly2385Arg is a common risk factor for PD in the Chinese population. Our bioinformatics and in-vitro studies also suggest that the Gly2385Arg variant is biologically relevant and it might act through pro-apoptotic mechanisms.

Aged↗
PubMed1998

Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2.

The chromosomal region of Pseudomonas sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes, styABCD, and two regulatory genes, stySR, were identified. This gene cluster when transferred to Escherichia coli W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source. Genes styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST. Northern blot analyses have confirmed that genes styABCD constitute a transcription unit. The transcription start site of the sty operon was mapped 33 nucleotides upstream of the styA translational start codon. The styS and styR genes, which form an independent transcriptional unit, are located upstream of the styABCD operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The styS gene product is homologous to histidine kinase proteins, whereas the styR gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains. Expression of the catabolic operon decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene, suggesting that the stySR system behaves as a styrene-inducible positive regulator of the styABCD operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene. This activity was found to be induced in Pseudomonas sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of Pseudomonas sp. strain Y2.

Amino Acid Sequence↗
PubMed2004

Comparative genetics of functional trinucleotide tandem repeats in humans and apes.

Several human neurodegenerative disorders are caused by the expansion of polymorphic trinucleotide repeat regions. Many of these loci are functional short tandem repeats (STRs) located in brain-expressed genes, and their study is thus relevant from both a medical and an evolutionary point of view. The aims of our study are to infer the comparative pattern of variation and evolution of this set of loci in order to show species-specific features in this group of STRs and on their potential for expansion (therefore, an insight into evolutionary medicine) and to unravel whether any human-specific feature may be identified in brain-expressed genes involved in human disease. We analyzed the variability of the normal range of seven expanding STR CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and two nonexpanding polymorphic CAG loci (KCNN3 and NCOA3) in humans, chimpanzees, gorillas, and orangutans. The study showed a general conservation of the repetitive tract and of the polymorphism in the four species and high heterogeneity among loci distributions. Humans present slightly larger alleles than the rest of species but a more relevant difference appears in variability levels: Humans are the species with the largest variance, although only for the expanding loci, suggesting a relationship between variability levels and expansion potential. The sequence analysis shows high levels of sequence conservation among species, a lack of correspondence between interruption patterns and variability levels, and signs of conservative selective pressure for some of the STR loci. Only two loci (SCA1 and SCA8) show a human specific distribution, with larger alleles than the rest of species. This could account, at the same time, for a human-specific trait and a predisposition to disease through expansion.

Analysis of Variance↗
PubMed1997

Early genetic and functional events in the pathogenesis of oral cancer.

Oral squamous cell carcinoma (SCC) is a major world health problem, but the changes leading to the development of malignancy remain essentially unknown. Early changes are thought to include the loss of tumour suppressor genes on chromosomes 3p, 9p, and 17p. Although what genes are involved on chromosome 3 remains speculative, p16 (9p21) and p53 (17p13) are inactivated in a high proportion of oral dysplastic lesions and carcinomas. SCC-derived cell lines are immortal, have decreased growth requirements in vitro, and show a variable capacity to form tumours in athymic mice. Normal oral keratinocytes and cells from potentially malignant lesions invariably senesce at early culture passage, have strict growth requirements in vitro, and are nontumorigenic in vivo. By contrast to normal oral keratinocytes, cells from potentially malignant lesions are defective in their capacity to terminally differentiate in suspension culture. Loss of cellular senescence and gain of the immortal phenotype is associated with inactivation of p16 and p53.

Animals↗
PubMed1977

Genetics and function of isocitrate lyase in Coprinus.

Thirteen chromosomal loci have been identified which affect acetate metabolism in Coprinus. Mutants at only two loci, acu-l and acu-7, are deficient in isocitrate lyase (ICL) (EC 4.1.3.1) activity. acu-1 mutants are unable to induce ICL because they lack acetyl-CoA synthetase which is required to convert acetate to the metabolic inducer of ICL. acu-7 is the structural gene for ICL. This was shown by selecting temperature sensitive acu+ revertants resulting from a second mutation within the acu-7 gene. One such revertant was shown to produce an ICL protein which was more thermolabile than the wild type enzyme. Other workers have postulated that ICL activity is important during asexual morphogenesis in fungi. No evidence was found for this in Coprinus. The morphological mutant oidial, which produces abundant asexual spores even in submerged culture, had the same low uninduced level of ICL activity as the wild type. Moreover, an acu-7 mutation had no effect on the expression of the oidial phenotype.

Acetates↗
PubMed1980

Genetic and functional analysis of tryptophan transport in Malpighian tubules of Drosophila.

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w+ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.

Animals↗
PubMed1991

The cytochrome c reductase/oxidase respiratory pathway of Paracoccus denitrificans: genetic and functional studies.

Data are presented on three components of the quinol oxidation branch of the Paracoccus respiratory chain: cytochrome c reductase, cytochrome c552, and the a-type terminal oxidase. Deletion mutants in the bc1 and the aa3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochrome c552 in electron transport between complex III and IV is presented.

Cytochrome c Group↗
PubMed1988

Hemolysin from Listeria--biochemistry, genetics and function in pathogenesis.

Thiol-activated hemolysins (listeriolysins) from Listeria monocytogenes (Sv4b) and Listeria ivanovii were purified to homogeneity. The N-terminal amino acid sequences of the 58 kDa listeriolysin of L. ivanovii and of a 24 kDa protein which may represent the CAMP-factor of L. ivanovii were determined. Antibodies raised against the L. ivanovii listeriolysin and anti-streptolysin O antibodies were used in Western blot analyses to detect listeriolysin(s) in virulent and avirulent Listeria strains. It was found that all virulent strains of L. monocytogenes synthesize and secrete listeriolysin (Mr 58-59 kDa), albeit in significantly variable quantities. No protein cross-reaction with anti-listeriolysin antibodies or anti-streptolysin O-antibodies was present in the supernatant of Listeria innocua, Listeria welshimeri, Listeria grayi and Listeria murrayi strains. Furthermore, the avirulent but hemolytic Listeria seeligeri did not cross-react with these antibodies. In a L. monocytogenes (strain EGD) gene bank constructed in Escherichia coli two types of hemolytic clones were identified. The first type carried recombinant plasmids with a common 2.0 kb fragment coding for a 23 kDa protein. This hemolytic activity was not activated by DTT and the 23 kDa protein did not cross react with anti-listeriolysin or anti-streptolysin antibodies. The other type of hemolytic clones was detected by using anti-streptolysin O antibodies to screen the gene bank. Some of these clones synthesized a protein of 61 kDa which cross reacted with anti-streptolysin O (or anti-listeriolysin) antibodies. By transposon Tn916 mutagenesis of L. monocytogenes two types of nonhemolytic mutants were obtained. Type I produced no extracellular protein that cross reacted with anti-listeriolysin (or anti SLO) antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗