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PubMed1992

Evolutionary relationships among the primate Mhc-DQA1 and DQA2 alleles.

The variation of the Mhc-DQA1 and DQA2 loci of ten different primate species (hominoids and Old World monkeys) was studied in order to obtain an insight in the processes that generate polymorphism of major histocompatibility complex (Mhc) class II genes and to establish the evolutionary relationships of their alleles. To that end nucleotide sequences of 36 Mhc class II DQA1 and seven DQA2 second exons were determined and phylogenetic trees that illustrate their evolutionary relationships were constructed. We demonstrate the existence of four primate Mhc-DQA1 allele lineages, two of which probably existed before the separation of the ancestors of the hominoids and Old World monkeys (approximately 22-28 million years ago). Mhc-DQA2 sequences were found only in the hominoid species and showed little diversity. We found no evidence for a major contribution of recombinational events to the generation of allelic diversity of the primate Mhc-DQA1 locus. Instead, our data suggest that the primate Mhc-DQA1 and DQA2 loci are relatively stable entities that mutated primarily as a result of point mutations.

Alleles↗

PubMed2002

Domain structure characterization of the multifunctional alpha-aminoadipate reductase from Penicillium chrysogenum by limited proteolysis. Activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain.

The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.

2-Aminoadipic Acid↗

PubMed1994

Protonic reactivity of sucrose in anhydrous hydrogen fluoride.

Sucrose reacts quantitatively, when dissolved at high concentration in anhydrous hydrogen fluoride, to afford a complex mixture of difructose dianhydrides and their glucosylated derivatives. Oligo- and small poly-saccharides up to dp 14 were detected by FABMS. Oligosaccharides up to dp 4, representing approximately 50% of the total mixture, have been isolated and characterized by mass spectrometry, 13C NMR spectroscopy, and comparison with reference oligosaccharides previously obtained by unambiguous synthesis. alpha-D-Fructofuranose beta-D-fructopyranose 1,2':2,1'-dianhydride is the main spirodioxanyl pseudodisaccharide entity found in the mixture, either free or glucosylated at C-6 and to a lesser extent at C-3, C-4, C-4', C-6, and C-5' C-6. Minor spirodioxanyl pseudodisaccharide components are di-beta-D-fructopyranose 1,2':2,1'-dianhydride, which has also been found glucosylated at C-5, alpha-D-fructopyranose beta-D-fructopyranose 1,2':2,1'-dianhydride, beta-D-fructofuranose beta-D-fructopyranose 1,2':2,3'-dianhydride, and the 6,6'-diglucosylated alpha-D-fructofuranose beta-D-fructofuranose 1,2':2,1'-dianhydride. A 13C NMR examination of the higher mass oligomeric fraction suggests that it may involve 6-O-isomaltooligoglycosyl alpha-D-fructofuranose beta-D-fructopyranose 1,2':2,1'-dianhydrides as the main structural components. The reaction of sucrose in anhydrous HF is believed to proceed through initial selective protonic activation of the tertiary anomeric carbon atom of the fructose moiety, resulting in the quantitative formation of difructose dianhydrides, which subsequently suffer electrophilic substitution by glucopyranosyl oxocarbenium ions generated in a second step by action of the HF.

Anhydrides↗

PubMed1999

Site-specific recombination at dif by Haemophilus influenzae XerC.

Xer site-specific recombination at the Escherichia coli chromosomal site dif converts chromosomal dimers to monomers, thereby allowing chromosome segregation during cell division. dif is located in the replication terminus region and binds the E. coli site-specific recombinases EcoXerC and EcoXerD. The Haemophilus influenzae Xer homologues, HinXerC and HinXerD, bind E. coli dif and exchange strands of dif Holliday junctions in vitro. Supercoiled dif sites are not recombined by EcoXerC and EcoXerD in vitro, possibly as a consequence of a regulatory process, which ensures that in vivo recombination at dif is confined to cells that can initiate cell division and contain dimeric chromosomes. In contrast, the combined action of HinXerC and EcoXerD supports in vitro recombination between supercoiled dif sites, thereby overcoming the barrier to dif recombination exhibited by EcoXerC and EcoXerD. The recombination products are catenated and knotted molecules, consistent with recombination occurring with synaptic complexes that have entrapped variable numbers of negative supercoils. Use of catalytically inactive recombinases provides support for a recombination pathway in which HinXerC-mediated strand exchange between directly repeated duplex dif sites generates a Holliday junction intermediate that is resolved by EcoXerD to catenated products. These can undergo a second recombination reaction to generate odd-noded knots.

Amino Acid Sequence↗

PubMed1993

Learning and alignment methods applied to protein structure prediction.

Learning techniques are able to extract structural knowledge specific to a selected set of proteins. We describe two algorithms that optimize scores expressing the propensity of a polypeptide sequence to adopt a local fold. The first algorithm generates secondary structure prediction rules based on a dictionary of geometrical patterns frequently found in the learning database. The second algorithm leads to scores that indicate the fit between an amino acid and a given local structural environment. Dynamic programming is then used to align structural information profiles by modifying the local mutation cost with the above learned functions. The main features of the system are exemplified on the structural prediction of the N-terminal domain of the CD4 antigen. Then the usefulness of additional 3-D information in the alignment is benchmarked on eight pairs of weakly homologous proteins.

Algorithms↗

PubMed1987

Simplified in vitro synthesis of mutated RNA molecules. An oligonucleotide promoter determines the initiation site of T7RNA polymerase on ss M13 phage DNA.

We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T7-oligo) which contains the T7RNA polymerase promoter sequence. In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g. M13 phage DNA) can be generated. The phage DNA represents the non-coding DNA strand for the desired transcript; the T7-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site. The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme. The reaction product is used directly as a template for T7RNA polymerase; cloning of mutants is not required.

Base Sequence↗

PubMed2005

DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.

Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.

Base Sequence↗

PubMed1985

Genes and pseudogenes for rat U3A and U3B small nuclear RNA.

We report here the isolation and primary structure of two genes encoding rat U3 small nuclear RNA. One of the genes encodes U3B RNA; the other encodes an RNA which is almost identical to U3A RNA. Both genes are expressed after microinjection into the nuclei of Xenopus laevis oocytes and can direct the accumulation of mature U3 RNA as well as longer transcripts which may be the U3 precursors. We have also isolated and sequenced four other regions of the rat genome homologous to U3 RNA. One of these almost certainly represents a second U3B gene; the other three are pseudogenes which appear to have been generated by the reverse flow of genetic information from U3 RNA back into the genome. Using genomic blotting techniques, we show that the rat U3 genes are present in only a few copies per haploid genome and are probably not closely linked to one another.

Animals↗

PubMed1992

Cloning and sequence analysis of two isotypic IgM heavy chain genes from Atlantic salmon, Salmo salar L.

A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.

Amino Acid Sequence↗

PubMed2001

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment.

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

Animals↗

PubMed1989

Infectivity of chimeric viroid transcripts reveals the presence of alternative processing sites in potato spindle tuber viroid.

In an investigation of viroid replication and pathogenesis, we have assessed the effect of sequence duplication of the upper central conserved region (CCR) of the molecule on the infectivity of RNAs transcribed in vitro from partial dimers of wild-type and mutant viroid cDNAs. In one set of experiments, the relative infectivities of one monomeric potato spindle tuber viroid (PSTV) and five oligomeric SP6 transcripts [PSTV or PSTV-TASV (tomato apical stunt viroid) chimeras] were compared. With one exception, the extent of sequence duplication in the CCR, and thus the length of the so-called palindrome, does correlate with an increase in specific infectivity. In a second set of experiments, in vitro generated site-specific mutations in cloned PSTV were used as markers to determine if a cleavage/ligation at sites other than the palindrome could generate infectious molecules in vivo. The creation of a novel PSTV-TPMV (tomato planta macho viroid) chimera in these experiments provides evidence that multimeric RNAs can be processed at sites other than the CCR to yield monomeric progeny.

Base Sequence↗

PubMed1980

Model for regulation of the histidine operon of Salmonella.

A model is proposed that accounts for regulation of the histidine operon by a mechanism involving alternative configuration of mRNA secondary structure (the alternative stem model). New evidence for the model includes sequence data on three regulatory mutations. The first (hisO1242) is a mutation that deletes sequences needed to form the attenuator mRNA stem and causes constitutive operon expression. The second mutation (hisO9654) is a His- ochre (UAA) mutation in the leader peptide gene; the existence of this mutation constitutes evidence that the leader peptide gene is translated. The third mutation (hisO9663) is remarkable. It neither generates a nonsense codon nor affects a translated sequence; yet, it is suppressible by amber suppressors. We believe this mutation causes a His- phenotype by interfering with mRNA secondary structure. The suppressibility of the mutation is probably due to disruption of the attenuator stem by ribosomes that read through the terminator codon of the leader peptide gene. This explanation is supported by the observation of derepression of a wild-type control region in the presence of an amber suppressor. Evidence is presented that hisT mutants (which lack pseudouridine in the anticodon arm of histidine tRNA) may cause derepression of the his operon by slowing protein synthesis in the leader peptide gene.

Bacterial Proteins↗

PubMed1998

EbEST: an automated tool using expressed sequence tags to delineate gene structure.

Large numbers of expressed sequence tags (ESTs) continue to fill public and private databases with partial cDNA sequences. However, using this huge amount of ESTs to facilitate gene finding in genomic sequence imposes a challenge, especially to wet-lab scientists who often have limited computing resources. In an effort to consolidate the information hidden in the vast number of ESTs into a readable and manageable format, we have developed EbEST-a program that automates the process of using ESTs to help delineate gene structure in long stretches of genomic sequence. The EbEST program consists of three functional modules-the first module separates homologous ESTs into clusters and identifies the most informative ESTs within each cluster; the second module uses the informative ESTs to perform gapped alignment and to predict the exon-intron boundary; and the third module generates text file and graphic outputs that illustrate the orientation, exonic structure, and untranslated regions (UTRs) of putative genes in the genomic sequence being analyzed. Evaluation of EbEST with 176 human genes from the ALLSEQ set indicated that it performed in-line with several existing gene finding programs, but was more tolerant to sequencing errors. Furthermore, when EbEST was challenged with query sequences that harbor more than one gene, it suffered only a slight drop in performance, whereas the performance of the other programs evaluated decreased more. EbEST may be used as a stand-alone tool to annotate human genomic sequences with EST-derived gene elements, or can be used in conjunction with computational gene-recognition programs to increase the accuracy of gene prediction. [EbBEST is available at http://EbEST.ifrc.mcw.edu]

Base Sequence↗

PubMed2003

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme.

We previously isolated from random sequences ribozymes able to form a glycosidic linkage between a ribose sugar and 4-thiouracil in a reaction that mimics protein-catalyzed nucleotide synthesis. Here we report on two serial in vitro selection experiments that defined the core motif of one of the nucleotide synthase ribozymes and provided improved versions of this ribozyme. The first selection experiment started from a degenerate sequence pool based on the previously isolated sequence and used a selection-amplification protocol that allowed the sequence requirements at the 3' terminus of the ribozyme to be interrogated. Comparing the active sequences identified in this experiment revealed the complicated secondary structure of the nucleotide synthase ribozyme. A second selection was then performed to remove nonessential sequence from the ribozyme. This selection started with a pool with variation introduced in both the sequence and the length of the nonconserved loops and joining regions. This pool was generated using a partial reblocking/deblocking strategy on a DNA synthesizer, allowing the combinatorial synthesis of both point deletions and point substitutions. The consensus ribozyme motif that emerged was an approximately 71 nt pseudoknot structure with five stems and two important joining segments. Comparative sequence analysis and a cross-linking experiment point to the probable location of nucleotide synthesis. The prototype isolate from the second selection was nearly 35 times more efficient than the initial isolate and at least 10(8) times more efficient than an upper limit of an as-yet undetectable uncatalyzed reaction, supporting the idea that RNA-catalyzed nucleotide synthesis might have been important in an RNA world.

Amino Acid Substitution↗

PubMed2002

Application of single strand conformational polymorphism (SSCP) analysis with fluorescent primers for differentiation of Schistosoma haematobium group species.

To assess the utility of single-stranded conformational polymorphism (SSCP) analysis for the differentiation of schistosomes, using methods adapted for a Perkin Elmer ABI Prism 377 automated sequencer, 3 isolates of Schistosoma haematobium, 2 of S. intercalatum and single isolates of S. curassoni and S. bovis were selected for study. Two fluorescently labelled, double-stranded polymerase chain reaction products, amplified from the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and the nuclear ribosomal second internal transcribed spacer (ITS2), were generated from single male and female worms. Changes in electrophoretic mobility of fragments within an SSCP profile revealed variation at individual, isolate and species levels. The mutational basis between representative SSCP profiles was confirmed by direct sequencing, demonstrating that single point substitutions were detectable. SSCP analysis has considerable potential as an alternative molecular method of identification and characterization of schistosomes. More broadly, fluorescence-based SSCP analysis is applicable to almost any gene target from any species of parasite and is a powerful molecular tool for genetic profiling.

Animals↗

PubMed1997

In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex.

Genetically simplified derivatives of complex retroviruses that replicate in animal models are useful tools to study the role of the complex regulatory genes in virus infection and pathogenesis and were proposed as a novel approach toward the development of vaccines against complex retroviruses. Previously we developed genetically simple derivatives of bovine leukemia virus (BLV) that can replicate in tissue culture independently of the BLV regulatory proteins, Tax and Rex, and the RIII and GIV open reading frames (K. Boris-Lawrie and H. M. Temin, J. Virol. 69:1920-1924, 1995). These derivatives are encoded on novel, hybrid retrovirus genomes that contain transcriptional control sequences of a simple retrovirus and gag-pol or env genes of the complex BLV. The first-generation simple BLV derivatives replicate as complementary viruses (coviruses) by using separate gag-pol or env genomes, and therefore virus spread is limited to cells that are infected with both covirus genomes. Here we describe a second-generation simple BLV derivative that is encoded on a single hybrid genome. We show the virus to be replication competent by successive passage on D17 target cells and by analysis of viral RNA and proteins in the infected cells. Furthermore, we evaluate the immunogenicity and infectivity of the simple BLV derivatives in a BLV animal model. Small groups of rats were injected either with virus-producing cells or with proviral DNA. Western immunoblot analysis revealed that antibodies against the major viral antigenic determinants are induced in response to either method of introduction and that seroconversion is sustained in most of the rats for at least 6 months (the duration of the study). The magnitudes of the antiviral responses were similar in rats infected with the first-generation simple BLV coviruses, the second-generation replication-competent derivative, or wild-type BLV. Wild-type BLV typically infects peripheral blood mononuclear cells (PBMC), and the simple BLV derivatives were also found to infect PBMC as demonstrated by PCR amplification of proviral sequences and reverse transcriptase PCR amplification of viral RNA in treated rats. These results establish that simple BLV derivatives lacking tax and rex are infectious and immunogenic in rats. These viruses will be useful tools in comparative studies with BLV to evaluate the role of tax and rex in maintenance of virus load and in disease outcome.

Animals↗

PubMed1996

Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes.

We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.

Adult↗

PubMed1992

Sequence and localization of human NASP: conservation of a Xenopus histone-binding protein.

In this study the sequence and localization of human testicular NASP (nuclear autoantigenic sperm protein) are reported. NASP cDNA contains 2561 nt encoding a protein of 787 amino acids. The open reading frame contains 2446 nt followed by an ochre stop codon (TAA) and 104 nucleotides of untranslated sequence containing a poly(A) addition signal 10 bases upstream of the poly(A) tail. Northern blot analysis of human testis poly(A) mRNA indicates a message of approximately 3.2 kb. Multiple sequence alignment (MSA) analysis of the encoded human NASP amino acid sequence with the sequence for the Xenopus histone-binding protein N1/N2 and the rabbit NASP amino acid sequence demonstrates that the human sequence and the Xenopus sequence have extensive amino acid homology upstream of the rabbit initiation codon. Significantly, there is an 85% identity between the human and the rabbit NASP sequences when the alignment starts at the N-terminal of the rabbit sequence and at amino acid 101 of the human sequence. The nuclear translocation signal found in N1/N2 and rabbit NASP is completely conserved in human NASP. The first histone-binding domain of Xenopus is 70% identical and 90% similar to the human NASP domain. The second histone-binding domain of Xenopus is 48% identical and 71% similar to the human NASP domain. MSA analysis of the three sequences generated an unrooted ancestral tree with two branches, indicating that fewer amino acid changes have occurred between the Xenopus and the human sequences than between the Xenopus and the rabbit sequences. In the human testis, NASP is localized predominantly in primary spermatocytes and round spermatids. Spermatogonia, Sertoli cells, Leydig cells, peritubular cells, and other somatic cells do not stain. Human spermatozoa contain NASP in the acrosomal region. Following the acrosome reaction, some NASP remains in the equatorial and postacrosomal regions. We propose that mammalian testes and sperm contain a histone-binding protein which may play a role in regulating the early events of spermatogenesis.

Amino Acid Sequence↗