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At least 163 records · Page 9Linked to original sources
PubMed2003

Mechanistic insights into oxidosqualene cyclizations through homology modeling.

2,3-Oxidosqualene cyclases (OSC) are key enzymes in sterol biosynthesis. They catalyze the stereoselective cyclization and skeletal rearrangement of (3S)-2,3-oxidosqualene to lanosterol in mammals and fungi and to cycloartenol in algae and higher plants. Sequence information and proposed mechanism of 2,3-oxidosqualene cyclases are closely related to those of squalene-hopene cyclases (SHC), which represent functional analogs of OSCs in bacteria. SHCs catalyze the cationic cyclization cascade converting the linear triterpene squalene to fused ring compounds called hopanoids. High stereoselectivity and precision of the skeletal rearrangements has aroused the interest of researchers for nearly half a century, and valuable data on studying mechanistic details in the complex enzyme-catalyzed cyclization cascade has been collected. Today, interest in cyclases is still unbroken, because OSCs became targets for the development of antifungal and hypocholesterolemic drugs. However, due to the large size and membrane-bound nature of OSCs, three-dimensional structural information is still not available, thus preventing a complete understanding of the atomic details of the catalytic mechanism. In this work, we discuss results gained from homology modeling of human OSC based on structural information of SHC from Alicyclobacillus acidocaldarius and propose a structural model of human OSC. The model is in accordance with previously performed experimental studies with mechanism-based suicide inhibitors and mutagenesis experiments with altered activity and product specificity. Structural insight should strongly stimulate structure-based design of antifungal or cholesterol-lowering drugs.

Amino Acid Sequence↗
PubMed1994

Polymerase chain reaction and sequencing for typing rhinovirus RNA.

Primers were designed and tested for their ability to distinguish rhinoviruses from enteroviruses. A primer set derived from the 5'-UTR/VP coding region junction was able to amplify all the rhinovirus serotypes tested. Enteroviruses were either not amplified by these primer pairs or produced a band of larger size that could easily be discriminated from the rhinovirus-specific product. In contrast, primers embedded in the 5'-UTR region alone were able to amplify both rhinovirus and enterovirus RNA. It is shown that rhinoviruses could be specifically typed by sequencing the amplicon derived from the 5'-UTR set. The sequences of the 5'-UTR region of ten previously unsequenced rhinoviruses were derived. The sequences obtained cluster into two groups: 1B, 41, 15, 30, 63, 31, 56, and 44; and 17, 69, and 70. Amplicons from serotypes 17,69, and 70 also group by sequence with the equivalent region of HRV14 from the genetic database, while the others group with 2 and 89.

Animals↗
PubMed2002

Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae.

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.

Base Sequence↗
PubMed2000

Searching genomes for sequences with the potential to form intrastrand triple helices.

The canonical double-helix form of DNA is thought to predominate both in dilute solution and in living cells. Sequence-dependent fluctuations in local DNA shape occur within the double helix. Besides these relatively modest variations in shape, more extreme and remarkable structures have been detected in which some bases become unpaired. Examples include unusual three-stranded structures such as H-DNA. Certain RNA and DNA strands can also fold onto themselves to form intrastrand triplexes. Although they have been extensively studied in vitro, it remains unknown whether nucleic acid triplexes play natural roles in cells. If natural nucleic acid triplexes were identified in cells, much could be learned by examining the formation, stabilization, and function of such structures. With these goals in mind, we adapted a pattern-recognition program to search genetic databases for a type of potential triplex structure whose presence in genomes has not been previously investigated. We term these sequences Potential Intrastrand Triplex (PIT) elements. The formation of an intrastrand triplex requires three consecutive sequence domains with appropriate symmetry along a single nucleic acid strand. It is remarkable that we discovered multiple copies of sequence elements with the potential to form one particular class of intrastrand triplexes in the fully sequenced genomes of several bacteria. We then focused on the characterization of the 25 copies of a particular approximately 37 nt PIT sequence detected in Escherichia coli. Through biochemical studies, we demonstrate that an isolated DNA strand from this family of E. coli PIT elements forms a stable intrastrand triplex at physiological temperature and pH in the presence of physiological concentrations of Mg(2+).

Algorithms↗
PubMed2004

Molecular mapping of resistance to Pyrenophora tritici-repentis race 5 and sensitivity to Ptr ToxB in wheat.

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease in the major wheat growing areas of the world. Multiple races of the pathogen have been characterized based on their ability to cause necrosis and/or chlorosis in differential wheat lines. Isolates of race 5 cause chlorosis only, and they produce a host-selective toxin designated Ptr ToxB that induces chlorosis when infiltrated into sensitive genotypes. The international Triticeae mapping initiative (ITMI) mapping population was used to identify genomic regions harboring QTLs for resistance to fungal inoculations of Ptr race 5 and to determine the chromosomal location of the gene conditioning sensitivity to Ptr ToxB. The toxin-insensitivity gene, which we are designating tsc2, mapped to the distal tip of the short arm of chromosome 2B. This gene was responsible for the effects of a major QTL associated with resistance to the race 5 fungus and accounted for 69% of the phenotypic variation. Additional minor QTLs were identified on the short arm of 2A, the long arm of 4A, and on the long arm of chromosome 2B. Together, the major QTL on 2BS identified by tsc2 and the QTL on 4AL explained 73% of the total phenotypic variation for resistance to Ptr race 5. The results of this research indicate that Ptr ToxB is a major virulence factor, and the markers closely linked to tsc2 and the 4A QTL should be useful for introgression of resistance into adapted germplasm.

Ascomycota↗
PubMed2000

An integrated web interface for large-scale characterization of sequence data.

Large-scale genome projects require the analysis of large amounts of raw data. This analysis often involves the application of a chain of biology-based programs. Many of these programs are difficult to operate because they are non-integrated, command-line driven, and platform-dependent. The problem is compounded when the number of data files involved is large, making navigation and status-tracking difficult. To demonstrate how this problem can be addressed, we have created a platform-independent Web front end that integrates a set of programs used in a genomic project analyzing gene function by transposon mutagenesis in Saccharomyces cerevisiae. In particular, these programs help define a large number of transposon insertion events within the yeast genome, identifying both the precise site of transposon insertion as well as potential open reading frames disrupted by this insertion event. Our Web interface facilitates this analysis by performing the following tasks. Firstly, it allows each of the analysis programs to be launched against multiple directories of data files. Secondly, it allows the user to view, download, and upload files generated by the programs. Thirdly, it indicates which sets of data directories have been processed by each program. Although designed specifically to aid in this project, our interface exemplifies a general approach by which independent software programs may be integrated into an efficient protocol for large-scale genomic data processing.

Clinical Laboratory Information Systems↗
PubMed1995

The influence of maternal weight on human chorionic gonadotropin in the multiple-marker screening test for fetal Down syndrome.

OBJECTIVE: Our purpose was to determine the effect of maternal weight on human chorionic gonadotropin concentration in the multiple-marker screening test for fetal Down syndrome. STUDY DESIGN: Two genetics databases were used: database I contained the results of 8297 multiple-marker screening tests and database II contained the results of 1936 multiple-marker screening tests and fetal karyotypes. RESULTS: The overall screen-positive rate in database I was 7.1%; it was 7.5% in patients weighing < 180 pounds and 5.1% in patients weighing > or = 180 pounds (p = 0.001). Weight significantly affected the screen-positive rate only in women > or = 30 years old (p = 0.003 for 30 to 34 years, p = 0.00004 for > or = 35 years). A weight correction formula was derived; when applied to database II it eliminated individual weight-related differences but had no effect on the overall screen-positive rate or Down syndrome detection rate. CONCLUSIONS: Human chorionic gonadotropin concentration is affected by maternal weight. A weight correction formula eliminates individual weight-related differences in the screen-positive rate but has no discernible effect on the overall screen-positive or Down syndrome detection rates.

Adult↗
PubMed1995

Primary structures of three Armenian hamster monoclonal antibodies specific for idiotopes and metatopes of the monoclonal anti-fluorescein antibody 4-4-20.

This paper reports the complete V gamma, V kappa, C gamma 1 and C kappa nucleotide and deduced amino acid sequences of two hamster monoclonal anti-metatype antibodies, 3A5-1 and 4A6. These antibodies have been previously characterized in terms of their binding and molecular stabilization properties with liganded murine monoclonal and single-chain antibody 4-4-20 active sites. Also reported are the complete V kappa and C kappa nucleotide and deduced amino acid sequence of hamster monoclonal anti-idiotype antibody 1F4, which is specific for the unliganded 4-4-20 active site. Oligonucleotide primers based on the 5' ends of murine variable genes, along with primers specific for murine IgG C gamma 1 and kappa constant region genes, have been used in cDNA and polymerase chain reactions (PCRs) to amplify IgG cDNA from Armenian hamster/mouse hybridomas. The hamster C gamma 1 and C kappa domain sequences are highly homologous to previously reported murine sequences. The anti-idiotype mAb V kappa gene demonstrated strong similarity to the murine V kappa V gene subgroup while the two anti-metatype mAb V kappa genes approximated more closely to the murine V kappa III gene subgroup. The two anti-metatype mAbs utilized highly homologous V gamma genes, with differing HCDR 3 regions, that appeared similar to the murine V gamma I(a) subgroup. These sequence determinations represent the first primary structures reported for antibodies with anti-metatype activity and are additions to the relatively sparse hamster immunoglobulin genetic database. Results are discussed in terms of 4-4-20 active site specificity and anti-metatype activity, as well as immunoglobulin structural diversity in an anti-Ig immune response.

Amino Acid Sequence↗
PubMed2004

Trypanosoma cruzi: cloning and characterization of two genes whose expression is up-regulated in metacyclic trypomastigotes.

The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative non-infective form of Trypanosoma cruzi into a non-replicative infective stage. The study of genes with stage-specific expression may provide insight into the mechanisms involved in the regulation of gene expression in this parasite. We cloned and characterized two genes whose expression is up-regulated in metacyclic trypomastigote, those encoding metacyclin-II (Met-II) and metacyclin-III (Met-III). Nucleotide sequence analysis identified no sequence similarity with sequences available from genetic databases. The deduced amino acid sequences of the genes indicated that Met-III encodes a basic polypeptide whereas Met-II encodes an acidic polypeptide. Northern and Western blot analyses showed that Met-II and Met-III were expressed by metacyclic trypomastigotes, but not by epimastigotes. Antisera directed against the recombinant Met-II and Met-III proteins recognized two polypeptides on Western blots: a 16-kDa and a 24-kDa polypeptide. Immunocytochemistry analysis using electron microscopy showed that metacyclin-II is localized mainly at the kinetoplast whereas metacyclin-III is localized at the nucleus of the parasite. Southern blot analysis, using genomic DNA and T. cruzi chromosomes separated by pulsed-field gel electrophoresis, indicated that these genes were present as single copies on different chromosomes of T. cruzi Dm28c.

Animals↗
PubMed2003

Integrating 'omic' information: a bridge between genomics and systems biology.

The availability of genome sequences for several organisms, including humans, and the resulting first-approximation lists of genes, have allowed a transition from molecular biology to 'modular biology'. In modular biology, biological processes of interest, or modules, are studied as complex systems of functionally interacting macromolecules. Functional genomic and proteomic ('omic') approaches can be helpful to accelerate the identification of the genes and gene products involved in particular modules, and to describe the functional relationships between them. However, the data emerging from individual omic approaches should be viewed with caution because of the occurrence of false-negative and false-positive results and because single annotations are not sufficient for an understanding of gene function. To increase the reliability of gene function annotation, multiple independent datasets need to be integrated. Here, we review the recent development of strategies for such integration and we argue that these will be important for a systems approach to modular biology.

Computational Biology↗
PubMed2026

Long-read Sequences Mapped to a Complete Reference Genome Uncover Uncaptured Structural Variants across the Beta-globin Cluster in Africans with Sickle Cell Disease.

African genomes are marked by extensive complexity in the number and distribution of variants, yet remain under-represented in genetic databases and the human reference genome. This gap in representation limits the broad application of genomic medicine. Sickle cell disease (SCD) - one of the most common monogenic diseases - has its highest prevalence in Africa, and variation in disease severity has consistently been linked to the beta-globin locus, including levels of fetal hemoglobin (HbF). Modulation of HbF is central to current SCD gene therapies; however, the inherent complexity and variation at the locus in African genomes presents a challenge to translating these advances to Africa. Here, we align long-read single molecule sequences (LRS) targeted to the beta-globin region to the hg38 and T2T-CHM13v2 genome references in 40 individuals with SCD, predominantly recruited from three African countries. We demonstrate that the expanded T2T-CHM13v2 reference sequence at this locus reduces Structural Variant (SV) calls by 70% and uncovers uncaptured single nucleotide variants (SNVs). Across the cluster we report 343 SVs and 196 SNVs that have not been previously reported, including in LRS data from the All of Us project. By including African populations from ethnolinguistic groups that have not been previously surveyed we improve variant resolution and bolster evidence for observed variation. Finally, we identify a common &#x223c;4kb insertion locus overlapping the HBB promoter among individuals with high HbF. These results demonstrate the utility of combining a comprehensive reference genome with LRS in African populations to uncover genomic variation at disease-associated loci.

SNV↗
PubMed2000

Ultrasound findings and multiple marker screening in trisomy 18.

OBJECTIVE: To compare detection of trisomy 18 in the second trimester by ultrasound and multiple-marker testing. METHODS: A computerized genetics database was used to identify fetuses of 14-22 weeks' gestation who had comprehensive ultrasound examinations, multiple-marker screening tests (alpha-fetoprotein [AFP]), hCG, unconjugated estriol [E3], and trisomy 18 karyotype. A positive trisomy 18 screen was defined as AFP up to 0.75 multiples of the median (MoM), hCG up to 0.55 MoM, and unconjugated E3 up to 0.60 MoM. A risk of at least 1:190 defined a positive Down syndrome screen. Ultrasound abnormalities were diagnosed prospectively and were confirmed later by retrospective review of sonographic images. RESULTS: From 1988-1997, 30 trisomy 18 fetuses who had comprehensive ultrasounds and multiple-marker testing were identified. Twenty-one (70%) had abnormalities detected by ultrasound, of which the most common isolated finding was choroid plexus cyst. Eleven fetuses (37%) had positive trisomy 18 screens, and two had positive Down syndrome screens, for a total of 13 of 30 (43%) fetuses with positive multiple-marker screening tests. CONCLUSION: We found that ultrasound was more likely to be abnormal than multiple-marker screening tests in fetuses with trisomy 18 (70%) (95% confidence interval [CI] 54, 86 versus 43% CI 25, 61). However, combining the two testing methods yielded the highest detection rate (80% [CI 66%, 94%]).

Adult↗
PubMed2002

The evolution of structural databases.

Starting with the Protein Data Bank (PDB) as a common ancestor, the evolution of structural databases has been driven by the rapprochement of the structural world and the practical applications. The result is an impressive number of secondary structural databases that is welcomed by structural biologists and bioinformaticians but runs the risk of producing an embarrassment of riches among non-specialist users. Given that any profit depends on the number of customers, efficient interfaces between many structural data banks must be available to make their contents easily accessible. Increasing the information content of central structural repositories might be the best way to guide users through the many, sometimes overlapping databases.

Computer Communication Networks↗
PubMed1998

PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired pneumonia. We identified a 73kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. Secretion occurred without cleavage of a signal peptide and in low-calcium environments in the presence of a divalent cation chelator, as is the case for characterized P. aeruginosa type III secreted proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino-terminal peptide sequence analysis indicated that the amino-terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A-X-K-S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5' of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5' of the PepA translational start site. Analysis of G + C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

Amino Acid Sequence↗