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PubMed2003

Spontaneous mutations restore the viability of tick-borne encephalitis virus mutants with large deletions in protein C.

The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these "double mutants" provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.

Amino Acid Sequence↗
PubMed2002

Diversity of Tn4001 transposition products: the flanking IS256 elements can form tandem dimers and IS circles.

We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.

DNA Transposable Elements↗
PubMed1997

Mutually exclusive splicing generates two distinct isoforms of pig heart succinyl-CoA synthetase.

We have identified two distinct cDNAs encoding the alpha-subunit of pig heart succinyl-CoA synthetase. The derived amino acid sequence of one of these, PHalpha57, is highly similar to the alpha-subunit of the rat liver precursor enzyme. The second cDNA, PHalpha108, was identical throughout its sequence with PHalpha57 except for a stretch of 108 nucleotides which replaced a 57 nucleotide sequence in PHalpha57. Coexpression of either alpha-subunit cDNA with a common pig heart beta-subunit cDNA produced isozymes with GTP-specific enzyme activity. The enzyme produced by the combination of PHalpha57 and the beta-subunit cDNA resembled the "native" enzyme purified from pig heart tissue. In contrast, the expressed enzyme from the combination with PHalpha108 was clearly distinguishable from the native enzyme by, for example, hydroxyapatite chromatography. Moreover, it was now apparent that this isoform had been observed in previous preparations of the native enzyme, but always in very low amounts and, thus, disregarded. We have shown further that the two mRNA transcripts arise from a single gene and are generated by mutually exclusive splicing. The production of the PHalpha108 message involves the use of a non-canonical splice site pair, AT-AA. Finally, we provide evidence for tissue specific regulation in the splicing of the PHalpha108 message.

Alternative Splicing↗
PubMed1998

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis.

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.

Amino Acid Sequence↗
PubMed2005

Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A).

PURPOSE: To describe the clinical features and genetic analysis of a family with an autosomal dominant cone dystrophy (adCD). METHODS: Selected members of a family with an autosomal dominant cone dystrophy underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and genomic fragments were amplified by PCR. Linkage to locus D6S1017 was established. DHPLC mutational analysis and direct sequencing were used to identify a mutation in GUCA1A, the gene encoding the guanylate cyclase activating protein 1 (GCAP1). RESULTS: Of 24 individuals who are at risk of the disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinogram performed on one proband revealed profoundly subnormal and prolonged photopic and flicker responses, but preserved scotopic ERGs, consistent with a cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a novel L151F mutation in GCAP1. Like the E155G mutation, this mutation occurs in the EF4 hand domain, a region of GCAP1 critical in conferring calcium sensitivity to the protein. The leucine at this position is highly conserved among vertebrate guanylate cyclase activating proteins. CONCLUSIONS: A novel L151F missense mutation in the EF4 high affinity Ca2+ binding site of GCAP1 is linked to adCD in a large pedigree. The cone dystrophy in this family shares clinical and electrophysiologic characteristics with other previously described adCD caused by mutations in GUCA1A.

Adult↗
PubMed2000

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases.

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.

Amino Acid Sequence↗
PubMed2007

Haplotype structure of FSHB, the beta-subunit gene for fertility-associated follicle-stimulating hormone: possible influence of balancing selection.

Follicle-stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor-binding beta-subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76-96.6% of each population's sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organism's fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.

Animals↗
PubMed2006

Rapid generation of new powdery mildew resistance genes after wheat domestication.

Plant defence against pathogens is controlled by disease resistance (R) gene products that directly or indirectly detect specific pathogen effectors. Plant-pathogen interactions have been proposed to follow a co-evolutionary arms-race model where R genes are recent and evolve rapidly in response to structural changes in matching pathogen effectors. However, the longevity and extensive polymorphism of R genes studied were more consistent with balancing selection maintaining ancient and diverse R genes or alleles. In bread wheat (Triticum aestivum), the Pm3 locus confers race-specific resistance to wheat powdery mildew (Blumeria graminis f.sp. triticii). Here we describe recently generated Pm3 resistance alleles that all derive from one susceptible allele, Pm3CS, which is widespread among hexaploid bread-wheat lines. One group of four Pm3 resistance alleles shows few, clearly delimited, polymorphic sequence blocks of ancient origin, embedded in sequences identical to Pm3CS and possibly derived from gene conversion. A second group of three alleles differs from Pm3CS by only two to five mutations, all non-synonymous, and all in the leucine-rich repeat-encoding region. Transient transformation experiments confirmed that Pm3 resistance specificities are based on one or few amino acid changes. The Pm3CS allele was found in wild tetraploid wheat, the ancestor of hexaploid bread wheat, specifically from southern Turkey, a region proposed to be the site of wheat domestication. Based on these data, we propose that the Pm3 resistance alleles were generated in agricultural ecosystems after domestication of wheat 10,000 years ago. The evolution of Pm3 alleles in wheat is best described by the model of evolved recycling, where novel genetic variation is integrated in plant populations together with recycling of old variation.

Alleles↗
PubMed2002

Emerging therapeutic targets in tuberculosis: post-genomic era.

Every minute, somewhere in the world four people die from tuberculosis (TB), yet it has been nearly 40 years since a novel drug was introduced to treat this disease. The ever increasing number of TB cases together with the advent of multi-drug resistant (MDR) TB, has stimulated the search for novel anti-TB agents. An array of novel drug targets is provided by the mycobacterial cell wall, whose integrity is essential for bacterial viability. Over the years researchers have identified potential drug targets that are associated with the synthesis of various cell wall constituents. This classic approach, together with the unravelling of the Mycobacterium tuberculosis genome sequence, has placed TB drug research in an unprecedented position. An entire new set of genetic and bioinformatic tools for probing potential drug targets is now available. As therapies using first-line drugs like isoniazid (INH) or rifampin in combination with second-line drugs, like ethambutol (EMB) still continues, a number of substituted fluoroquinolones are being considered as the new generation of anti-TB drugs for their favourable pharmacokinetic profile and excellent oral bioavailability. In this review, the future of anti-TB drugs is discussed with reflection on the structure and biosynthesis of cell wall constituents that are potential drug targets. The importance and relevance of the M. tuberculosis genome sequence for the development of novel anti-TB drugs, have also been underscored.

Animals↗
PubMed2000

Physical mapping of bchG, orf427, and orf177 in the photosynthesis gene cluster of Rhodobacter sphaeroides: functional assignment of the bacteriochlorophyll synthetase gene.

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999). An operonal arrangement of the genes is proposed.

Amino Acid Sequence↗
PubMed1992

Three genes for the human high affinity Fc receptor for IgG (Fc gamma RI) encode four distinct transcription products.

Three distinct but closely related classes of receptors that bind the Fc portion of immunoglobulin G (Fc gamma RI, -II, and -III) have been identified in humans. Only Fc gamma RI has high affinity for ligand and has a unique third extracellular domain (EC3). We have characterized three genes for human Fc gamma RI (A, B, and C). Each gene consists of six exons, spans 9.4 kilobase pairs, and localizes to chromosome 1. Although they are remarkably similar, genes B and C are notably different from A; in-frame stop codons are present in the EC3 domain of genes B and C, and deletions occur in a splice donor sequence of gene B. Four distinct Fc gamma RI transcripts were analyzed. One transcript, from gene A, would encode a transmembrane receptor with three external domains. A second transcript, an alternatively spliced product of gene B, would encode a two-external domain transmembrane receptor. Two transcripts, from genes B and C, have stop codons in EC3 and would be predicted to generate secreted receptors.

Amino Acid Sequence↗
PubMed2004

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes.

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)-BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Animals↗
PubMed2000

The orf162b sequence of Rhodobacter capsulatus encodes a protein required for optimal levels of photosynthetic pigment-protein complexes.

The orf162b sequence, the second open reading frame 3' of the reaction center (RC) H protein gene puhA in the Rhodobacter capsulatus photosynthesis gene cluster, is shown to be transcribed from a promoter located 5' of puhA. A nonpolar mutation of orf162b was generated by replacing most of the coding region with an antibiotic resistance cartridge. Although the mutant strain initiated rapid photosynthetic growth, growth slowed progressively and cultures often entered a pseudostationary phase. The amounts of the RC and light harvesting complex I (LHI) in cells obtained from such photosynthetic cultures were abnormally low, but these deficiencies were less severe when the mutant was grown to a pseudostationary phase induced by low aeration in the absence of illumination. The orf162b mutation did not significantly affect the expression of a pufB::lacZ translationally in-frame gene fusion under the control of the puf promoter, indicating normal transcription and translation of RC and LHI genes. Spontaneous secondary mutations in the strain with the orf162b disruption resulted in a bypass of the photosynthetic growth retardation and reduced the level of light harvesting complex II. These results and the presence of sequences similar to orf162b in other species indicate that the Orf162b protein is required for normal levels of the photosynthetic apparatus in purple photosynthetic bacteria.

Artificial Gene Fusion↗
PubMed1993

Diversification, not use, of the immunoglobulin VH gene repertoire is restricted in DiGeorge syndrome.

Immunoglobulin (Ig) genes were isolated from unamplified conventional as well as polymerase chain reaction-generated cDNA libraries constructed from the peripheral blood cells of a patient with complete DiGeorge syndrome. Comparison of the sequences of 36 heavy chain clones to the recently expanded database of human VH genes permitted identification of the germline VH genes that are expressed in this patient as well as placement of 19 of these genes in a partially resolved 0.8-mb region of the human VH locus. The pattern of VH gene use does not resemble the fetal (early) repertoire. However, as in the fetal repertoire, there are a number of cDNAs derived from germline genes that previously have been identified as autoantibodies. Two D mu sequences also were identified, as was another sequence resulting from a unique recombination event linking JH to an unidentified sequence containing a recombination signal sequence-like heptamer. All of the DiGeorge cDNAs are closely related to germline VH genes, showing little or no evidence of somatic mutation. In contrast, comparably selected IgM VH sequences derived from normal adult and age-matched human libraries, and from a second DiGeorge syndrome patient in whom the degree of thymic dysfunction is much less severe, exhibit considerable evidence of somatic mutation. The absence of somatic mutation is consistent with the atypical development of functional antibody responses associated with complete DiGeorge syndrome and implicates a role for T cells in the generation of diversity within the B cell repertoire.

Antibody Diversity↗
PubMed1996

Identification of Plasmodium falciparum MSP-1 peptides able to bind to human red blood cells.

To determine amino acid sequences of the Plasmodium falciparum MSP-1 protein that interact with red blood cell membranes in a specific receptor-ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP-1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP-1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide-RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intraerthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide-based malaria vaccines.

Amino Acid Sequence↗
PubMed1990

Organization and expression of the Drosophila FMRFamide-related prohormone gene.

The Drosophila FMRFamide gene is comprised of two exons separated by a 2.8-kb intervening sequence. The first exon consists of the 5' untranslated region and is spliced to the initiator methionine codon. The second exon encodes the prohormone and the 3' untranslated region of the mRNA. The promoter region contains a TATA box 30 nucleotides upstream from a consensus transcription start site. Immunohistochemical studies using antibodies generated against synthetic peptides and in situ hybridization histochemistry define a set of about 40 neurons in the brain and ventral ganglia that express the prohormone gene.

Amino Acid Sequence↗
PubMed1995

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Animals↗