Changes In Amino Acid Sequence Research Articles

Karakterisasi Molekular Fragmen Gen mexB Isolat Pseudomonas aeruginosa Multiresisten

Antibiotics have been widely used in the treatment of infectious diseases. However, their effectiveness has been questioned due to the tendency of some bacterial resistance to antibiotics. Pseudomonas aeruginosa among others has been known to be resistant to several antibiotics due to its MexABOprM efflux pump. Perhaps, the nucleotide sequence of its mexB gene fragment has changed followed by changes in amino acid sequence leading to alteration of the substrate recognition site. This alteration causes disability of antibiotics to recognize it and they are pumped out from the bacterial cell causing decrease in its inhibition concentration. An observasional study was performed using four P. aeruginosa isolates (A,B,C and D) taken from four laboratories in Bandung, and the sensitivity test for several antibiotics (tetracyclin, ampicyllin, amoxicyllin-clavulanat, kanamycin, ciprofloxazin, trimetoprim-sulphametoxazol, chloramphenicol dan eritromycin), was performed using Kirby-Bauer method. The Minimum Inhibitory Concentrations (MICs) for 4 isolates were 20.57-39.07 mg/ml for erithromycin, 29.35-48.57 mg/ml for kanamycin, 30.35-68.75 mg/ml for tetracyclin, 45.57-97.50 mg/ml for ampicyllin, 23.69-97.50 mg/ml for chloramphenicol, 25.82-59.56 mg/ml for amoxcyllinclavulanat,21.88-79.00 mg/ml for trimetoprim sulphametoxazol, and 20.58-56.97 mg/ml for ciprofloxazin. The increasing of MIC to each antibiotic was shown for 4 isolates of P. aeruginosa multiresistant to several antibiotics being studied. PCR technique was used to detect mexB gene fragment asumed as the substrate recognition site. The percentage of homology between the nucleotide sequence of mexB multiresistant P. aeruginosa and mexB P. aeruginosa producing siderophore pioverdin (Acc. No. L11616, NCBI) showed 96%, 100%, 97%, and 96% homology for P. aeruginosa A,B,C and D respectively. Employing DNAstar program, fragment variant of mexB gene of 4 multiresistant isolates A, B, C and D was detected. This variation lead to amino acid substitution of Gly-417->Ser, Glu-417->Gln, Thr-424->Pro, Tyr-328->Phe, Asp-328->His for P. aeruginosa A,B,C and D respectively, along with the change of their secondary structure, that changed the functional protein of MexB.

Evolving genetic code

In 1985, we reported that a bacterium, Mycoplasma capricolum, used a deviant genetic code, namely UGA, a “universal” stop codon, was read as tryptophan. This finding, together with the deviant nuclear genetic codes in not a few organisms and a number of mitochondria, shows that the genetic code is not universal, and is in a state of evolution. To account for the changes in codon meanings, we proposed the codon capture theory stating that all the code changes are non-disruptive without accompanied changes of amino acid sequences of proteins. Supporting evidence for the theory is presented in this review. A possible evolutionary process from the ancient to the present-day genetic code is also discussed.

ISOFORMS OF LITOPENAEUS VANNAMEI ANTI-LIPOPOLYSACCHARIDE AND ITS EXPRESSION BY BACTERIAL CHALLENGE

Anti-lipopolysaccharide factors have been described in arthropods, including penaeid shrimp. In this study, we characterize six antilipopolysaccharide-coding clones isolated from a cDNA library from L. vannamei hemocytes. Three different isoforms (named ALFLv1, ALFLv2 and ALFLv3) are observed, based on three nucleotide differences that produce three changes in amino acid sequence (Val11Ala, Val12Ala, Arg83Lys). Other single differences in nucleotide sequences were also noted, but they do not change the translated product. Considering that the signal peptide cleavage site occurs between Ala25 and Gln26, the substitutions Val11Ala and Val12Ala are not present in the mature protein. The mRNA steady state levels of ALFLv3, but not for ALFLv1 or ALFLv2, were up regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid ALFs seem to be associated with the Arg83Lys substitution and its expression under bacterial inoculation.

Sensitivity of FCV to recombinant feline interferon (rFeIFN)

Feline calicivirus cause feline respiratory diseases, and inactivated and attenuated vaccines are available for its prevention. Moreover, the presence of vaccine breakdown strains (VBS) is problematic. In Japan, feline recombinant interferon (rFeIFN) has been used for its treatment. However the method of compare with each strains has not established. To examine the relationship between the breakdown vaccine strain and rFeIFN sensitivity, the sensitivity of 47 field isolates to rFeIFN was determined. The Log PDD(50) values were normally distributed within the range 1.1-3.7, with a mean value of 2.3 +/- 0.64. Since 68.3% of the PDD values fell in the range of the mean +/- standard deviation, the values in the range 1.7-2.9, the lower values, and the higher values were defined as representing moderate, low, and high sensitivity, respectively. Among the 15 vaccine breakdown strains, strain Fukuoka9 showed a low sensitivity, but strains ML89, T58, and N74 were highly sensitive, showing no association with vaccine breakdown. The amino acid sequence changes specific to the low rFeIFN-sensitive Fukuoka-9 strain were found, suggesting that these sites are involved in rFeIFN sensitivity.

The role of human leukocyte antigen E and G in HIV infection

The role of human leukocyte antigen E and G in HIV infection

Replacement of Nonmuscle Myosin II-B with II-A Rescues Brain but Not Cardiac Defects in Mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B(-)/B(-) mice, most B(a*)/B(a*) mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B(-)/B(-) mice, 15 B(a*)/B(a*) mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the B(a*)/B(a*) mice showed evidence for hydrocephalus that is always found in B(-)/B(-) mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in B(a*)/B(a*) mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.

False Positive Results of Mitochondrial DNA Depletion/Deletion due to Single Nucleotide Substitutions Causing Appearance of Additional PvuII Restriction Sites

Human mitochondrial diseases are usually caused by dysfunction of mitochondrial DNA (mtDNA), particularly by point mutations, deletions, or depletions. In commonly used procedures for molecular diagnostics of mitochondrial dysfunction, one of the first steps is linearization of circular mitochondrial genomes with either BamHI or PvuII restriction endonulease, which cuts human mtDNA at a unique site. Here, we describe a case of false positive results, which suggested mtDNA depletion or a large deletion in a patient's tissue sample. More detailed analysis (mtDNA sequencing) revealed that these false positive results were caused by the presence of the 12753A>G substitution in the gene coding for NADH dehydrogenase subunit 5 (ND5). This substitution results in no change in amino acid sequence of the gene product but creates an additional PvuII site. Investigating a population of 200 patients not affected by mitochondrial diseases, we found an additional case of 12753A>G, and also another substitution, 12804T>C, which also results in no change in amino acid sequence of ND5 but creates an additional PvuII site. A few cases of 12753A>G and 12804T>C substitutions were found previously in Asian, American, African, and European populations (though they were not reported to date in the MITOMAP), but those samples were used in population studies and not tested for mtDNA deletion or depletion. Therefore, we present a cautionary report indicating that these mtDNA polymorphisms exist in various human populations (and thus, they are panethnic) and may cause false positive results of standard molecular analyses, including molecular diagnostics, of human mtDNA.

Genetic association of DLG5 variant R30Q with IBD in a familial inflammatory bowel disease (IBD) registry

The gene DLG5, a member of membrane associated guanylate kinase family, contains multiple functional domains such as leucine zipper, PDZ1, PDZ2, SH3, and GUK domains distributed over the entire gene. Any genetic variant that affects the structure of these domains may have impact on protein function in cell junction, maintenance of cell shape or clustering of channel proteins at the cell surface. A possible role of genetic variants of DLG5, such as R30Q, G1066G, e 26, and P1371Q, has been studied in IBD. However, for a better understanding of the contribution of these variants to IBD, more studies are needed. Using a central Pennsylvania familial IBD registry, we studied the genetic association of DLG5 with IBD. In addition to our DLG5 R30Q study reported elsewhere, we also analyzed four other single nucleotide polymorphisms (SNPs). SNPs E514Q, P979L, and P1371Q, are missense mutations that lead to protein amino acid sequence changes. SNP G1066G is at the second nucleotide of 5′ end of exon 16. Although it does not change the coding amino acid, it may have impact on DLG5 RNA splicing. A total of 244 individuals from 58 families, in which at least 2 individuals in each family were affected with IBD, were studied. 182 DNA samples from individuals without IBD served as controls. Genomic DNA isolated from lymphocyte and tissues was used for genotyping. The genotype analysis was carried out with PCR-based nature restriction fragment length polymorphism (RFLP) and converted RFLP methods. We found that: 1) for SNP G1066G, even the minor allele T is distributed to almost half of the population with no difference between diseased and nondiseased individuals. 2) Both E514Q and P979L are rare mutations. The frequency of mutated allele carrier is 4.5% for E514Q and 2.9% for P979L. The two mutations exist in both IBD patients and controls, their association with IBD is currently unclear due to the lack of statistic power. For SNP P1371Q, the frequency of mutation allele A is 8.2% in 244 individuals of the familial registry, and 11% in the 112 IBD affected individuals of the familial registry, and 9% in 169 sporadic IBD patients from our tissue bank, while only 6% in 182 normal controls. The results described above indicate that the DLG5 variant G1066G is probably not associated with IBD, whereas the variant P1371Q isfound at an increased incidence in both sporadic and familial form of IBD. The association of DLG variants, E514Q and P979L with IBD is currently unclear.

Creating Retroviral Vector Encoding Two Homologous Chimeric Immune Receptors with Degenerated Codons (47.28)

Abstract T cells genetically modified to express chimeric immune receptors (CIR) have been shown to be promising for immunotherapy. T cell receptor (TCR)-based CIR can be created in that the extracellular domains (Ec) of alpha (a) and beta (b) chains are fused to signaling cytoplasmic domain (Cy) of CD3 zeta (z) (TCRa-z and TCRb-z). To reduce homologous recombination associated with high homology between two nucleotide sequences in the same vector, we applied a novel approach by substituting the nucleotide sequence of one of the two homologous genes with degenerated genetic codes without change in amino acid sequence. In this study, we created a mutated DNA sequence encoding the Cy of z with degenerated codons (muz). A retroviral vector encoding a pair of TCR based CIRs (TCRa-z and TCRb-muz; tcsv-p36z) was created, with specificity via the pair of Ec of TCR a/b to a JC virus peptide p36/HLA-A2. 293T cells were transduced with retrovirus from PG13 cells with the tcsv-p36z. The transduced cells that expressed substantial TCR Vb of the CIR were able to bind p36/A2 tetramer, indicating the expression of both a and b chains in the single cell level. Western blot confirmed that both a and b chains were cellularly expressed intact. This approach, which we employed in creating retroviral vector encoding tc-CIRs with high homology, can be universally applied to creating viral vectors encoding two or multiple homologous molecules for gene therapy.

Investigation of hHB genes in a predigree of congenital monilethrix

Objective To investigate the gene polymorphism in a pedigree of congenital monilethrix. Methods Genomic DNA of affected members, the normal members of the pedigree and 50 unrelated normal members who came from different regions were extracted with a whole blood genomic DNA extraction kit and used as a template for the polymerase chain reaction (PCR)-mediated amplification of hHB1 and hHB6 genes. Results In the pedigree. DNA analysis of patients and normal persons revealed C(447th) in exonl of hHB1 gene and the 52th codon was CCA encoding arginine. But it was a heteropeak of G or C in 50 unrelated normal members, which encodes glycine or arginine. It showed that this change was a single nucleotide polymorphisms (SNP). Conclusion A genetic heterogeneity of monilethrix exists in Chinese population. SNP which can result in the change of amino acid sequence is found in a pedigree of congenital monilethrix, and a genetic heterogeneity of monilethrix existed in Chinese population.

Tissue-specific expression and molecular modeling of cytosolic manganese superoxide dismutases from the white shrimp Litopenaeus vannamei

Manganese superoxide dismutases (MnSODs) are usually mitochondrial enzymes, although there are few examples of cytosolic MnSODs (cMnSOD). We have previously characterized a cMnSOD cDNA from Litopenaeus vannamei hemocytes, and to obtain new insights into the tissue specific expression and the protein structure, we characterized three more different cMnSOD transcripts (cMnsod1, cMnsod2 and cMnsod3) and modeled the three-dimensional protein structure using human MnSOD as a template. The nucleotide sequences differ in seven positions. Four differences are silent; while three produce changes in amino acid sequence. cMnsod1, cMnsod2 and cMnsod3 are differentially expressed in nervous system, hepatopancreas and hemocytes. The structural protein model predicts bona fide MnSODs with proper coordination for the enzymatic activity.

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure

Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

The Histone Variant Macro-H2A Preferentially Forms “Hybrid Nucleosomes”

The histone domain of macro-H2A, which constitutes the N-terminal one third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant containing nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes containing one chain each of major H2A and macro-HA in vitro. The 2.9-A crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-containing nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.

A granule cell neuron-associated JC virus variant has a unique deletion in the VP1 gene

The human polyomavirus JC (JCV) typically infects glial cells and is the aetiological agent of progressive multifocal leukoencephalopathy (PML), which occurs in immunosuppressed individuals. The full-length sequence of a granule cell neuron-tropic JCV variant, JCV(GCN1), associated with lytic infection of granule cell neurons and cerebellar atrophy in a human immunodeficiency virus-infected patient with PML was determined and compared with the sequence of the JCV isolate from the classic PML lesions present in the hemispheric white matter of the same individual (JCV(HWM)). A unique deletion was found in the C terminus of the VP1 gene of JCV(GCN1), which encodes the major capsid protein, resulting in a frame shift and a total change of the C-terminal amino acid sequence of this protein. This deletion was not present in JCV(HWM), suggesting that this mutation may be instrumental in facilitating entry or replication of JCV into granule cell neurons.

Characterization of a novel DRB1*14 allele (DRB1*1449) in the Han‐Chinese population

We report here a novel human leukocyte antigen (HLA) allele, DRB1*1449, in the Han-Chinese population. The nature of the new allele was confirmed by the sequencing-based typing (SBT) method. Genomic DNA and six subclones containing DNA fragment of DRB1 exon 2 were sequenced in both forward and reverse directions. The exon 2 nucleotide sequence of DRB1*1449 is closely related to DRB1*1432 allele based on sequence homology. It has four nucleotide (nt) substitutions at positions 71, 196, 244 and 245 in exon 2, which lead to changes of amino acid sequences. The serological assignment of DRB1*1449 is DR14 based on the serological HLA typing result. This novel allele might be a result of recombination between DRB1*1402 and DRB1*140101 like alleles, which are very common among Chinese population.

Cooperativity Between Different Nutrient Receptors in Germination of Spores of Bacillus subtilis and Reduction of This Cooperativity by Alterations in the GerB Receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.

PIK3CA mutations in nasopharyngeal carcinoma

<i>PIK3CA</i> mutations in nasopharyngeal carcinoma

Variants in candidate ALS modifier genes linked to Cu/Zn superoxide dismutase do not explain divergent survival phenotypes

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). In North America, SOD1 A4V is the most common SOD1 mutation. A4V ALS cases typically have a very short survival (1–1.5 years versus 3–5 years for other dominant SOD1 mutations). A recent study of A4V carriers identified a common haplotype around the SOD1 locus, suggesting the hypothesis that genetic variations within the haplotypic region might accelerate the course of A4V cases. By contrast, SOD1 D90A/D90A ALS cases have a very slow progression (>10 years), raising the reciprocal hypothesis that modifier genes linked to SOD1 ameliorate the phenotype of recessively inherited SOD1 D90A/D90A mutations. In the present study, DNA sequencing of four genes within the haplotypic region shared in A4V and D90A ALS patients revealed 15 novel variants, but none result in changes in amino acid sequences specifically associated with SOD1 D90A/D90A or SOD1 A4V ALS. We conclude that mutations within coding regions of genes around the SOD1 locus are not responsible for the more aggressive and more benign natures of the SOD1 A4V and SOD1 D90A/D90A mutations, respectively.

Genetic predictors for drug resistance in soft tissue sarcoma: a review of publications in 2004

Sarcomas are generally managed through a multidisciplinary approach including surgery, radiotherapy, and chemotherapy. Metastatic tumor cells frequently develop resistance to cytotoxic agents. Several molecular mechanisms of drug resistance have been uncovered recently. Among genes governing the apoptosis pathway, overexpression of the bcl2 family or mutations of p53 have recently been reported to be involved in drug resistance in sarcoma. The multidrug resistance genes are involved in the efflux of chemotherapeutic agents. Identification of members of this family of genes may predict resistance for developing new strategies. Several histologic subtypes of sarcomas have specific mechanisms of resistance. Methotrexate is one of the four active drugs for osteosarcoma and penetrates in tumor cell through specific carrier (reduced folate carrier) whose levels of expression or changes in amino acid sequence correlate to resistance to methotrexate. Specific molecular alteration defining nosological entities among sarcoma also correlates to drug resistance. Thirty percent of human sarcomas are characterized by specific translocations that may predict, for specific subtypes, survival and response to treatment. Finally, gastrointestinal tumors harbor specific activating mutations in KIT or PDGFRA genes, which are responsive to imatinib. Specific mutations of the KIT and PDGFRA genes have now repeatedly been found correlated to response or resistance to imatinib. The molecular alterations present in tumor cells predict influence sensitivity or resistance to chemotherapy. Ultimately, this may delineate specific therapeutic strategies for both cytotoxic and targeted therapies in sarcomas.

Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.