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PubMed1989

Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. A further study of alterations in the glycophorin C gene.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5' and 3' flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.

Base Sequence↗
PubMed1988

The Z family, a group of transposed human immunoglobulin V kappa genes.

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb. The Z regions show extensive homology to each other according to restriction maps and hybridization data. In each Z region a solitary V kappa I gene was found. No V kappa genes of other subgroups were detected by hybridization. The nucleotide sequence of the ZI gene revealed a non-processed V kappa I pseudogene. Hybridization experiments with DNAs from rodent/human cell hybrids and other experimental data indicate that some and possibly all members of the Z family lie outside of the kappa locus which is located on chromosome 2; they have been transposed to other chromosomes. Because of their separation from the J kappa C kappa gene segment, the Z genes can be classified as pseudogenes independent of their sequences. We postulate that the Z family arose by amplification event(s). The Z regions can also be regarded as a small family of very long repetitive sequences.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the zucchini yellow mosaic virus capsid-encoding gene and its expression in Escherichia coli.

Zucchini yellow mosaic virus (ZYMV) RNA was purified and used as a template for the synthesis of cDNA. A partial restriction map covering 9.4 kb of the ZYMV genome was constructed from three clones designated ZYKS-22, ZYKS-16 and ZYKS-3. Sequencing the 3'-end region of the ZYMV genome indicates the presence of (A)48 chain. This is followed by an untranslated region of 210 nucleotides (nt) and a coding region of 837 nt corresponding to the putative virus coat protein (Cp) gene (cp). The predicted amino acid (aa) sequence of Cp derived from the cDNA showed about 50% to 62% homology with the known aa sequence for Cp of six other potyviruses. A construct of the putative cp was subcloned in frame with the lacZp gene promoter in a Bluescript plasmid and expressed in Escherichia coli cells. The fusion polypeptides (34 and 41 kDa), positively reacted in Western blots with an antiserum prepared against the native virus Cp.

Amino Acid Sequence↗
PubMed1994

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Animals↗
PubMed1994

Genetic variability of the beta-tubulin genes in benzimidazole-susceptible and -resistant strains of Haemonchus contortus.

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding beta-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two beta-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.

Alleles↗
PubMed1992

A nuclear gene encoding beta-amylase of sweet potato.

A nuclear AmyB gene from sweet potato encoding beta-amylase (beta Amy) that is abundant in tuberous roots and inducible in other organs by an exogenous supply of sucrose or polygalacturonic acid, was isolated and characterized. Genomic Southern blot hybridization, restriction maps of independently isolated phage lambda genomic clones, and the nucleotide sequence of AmyB compared with that of the cDNA, all suggested that beta Amy of sweet potato is encoded by a gene that is present in a single copy per haploid genome. In the sequence of AmyB, the sequence that is identical to that of the cDNA was split into seven exons by six introns, and the transcription of this gene starts from multiple sites 26 to 30 bp downstream from a potential TATA-box sequence, 5'-TATATAA. In the 5'-upstream region of AmyB, there are sequences homologous to those conserved in the 5'-upstream regions of genes encoding sporamin, which are regulated similarly to AmyB. The 5'-upstream region of AmyB also contains sequences to which several previously known plant nuclear factors bind.

Amino Acid Sequence↗
PubMed1993

Structure, expression and chromosomal localization of the gene encoding human 2',3'-cyclic-nucleotide 3'-phosphodiesterase.

Four human genomic DNA clones for 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) were isolated by screening a human genomic library with human CNP cDNA clones as probes. Restriction mapping and sequence analysis revealed that the human CNP gene is about 8.5 kb long and composed of four exons interrupted by three introns. There are two transcription start points and in human brain, two forms of CNP mRNA are produced from a single gene by alternative splicing, similar to mouse. A homology search of the 5'-flanking regions of exon 0 and exon 1 in the human CNP gene indicated the presence of oligodendroglia-specific elements and myelin basic protein transcription element (MBTE) motif, in addition to TATA-box-like sequences. Spot blot hybridization of flow-sorted human chromosomes with the 3'-noncoding region of the human CNP cDNA showed the localization of CNP to chromosome 17.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1989

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1.

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.

Animals↗
PubMed1992

Isolation and partial characterization of infectious molecular clones of feline immunodeficiency virus obtained directly from bone marrow DNA of a naturally infected cat.

Replication-competent molecular clones of feline immunodeficiency virus (FIV) were isolated directly from the DNA of bone marrow cells of a naturally FIV-infected cat. After transfection in a feline kidney cell line (CrFK) and subsequent cocultivation with peripheral blood mononuclear cells (PBMC), the viral progeny of the clones was infectious for PBMC but not for CrFK cells. PBMC infected with these clones showed syncytium formation, a decrease in cell viability, and gradual loss of CD4+ cells. The restriction maps of these clones differed from those obtained for previously described molecular clones of FIV derived from cats in the United States. The predicted amino acid sequence similarity of the envelope genes of the two clones was 99.3%, whereas the similarities of the sequences of the clones to those of two molecular clones from the United States, Petaluma and PPR, were 86 and 88%, respectively. Most of the differences between the amino acid sequences of the two clones and those of the clones from the United States were found in five different hypervariable (HV) regions, HV-1 through HV-5. The viral progeny of one of these clones was inoculated into two specific-pathogen-free cats. The animals seroconverted, and the virus could be reisolated from their PBMC.

Amino Acid Sequence↗
PubMed1992

Construction of a genomic DNA library of the Toxoplasma gondii ZS2 strain, screening of specific clones, and DNA diagnosis of toxoplasmosis.

We have constructed a genomic DNA library of the Toxoplasma gondii ZS2 strain and isolated a specific cloned DNA sequence from this organism. The restriction map of this cloned 1.1-kb DNA fragment was analyzed. Southern and dot-blot analyses showed that the 32P-labeled DNA fragment hybridized to parasite DNA, to DNAs from peripheral blood leukocytes and the thymus of baby pigs that were artificially infected with T. gondii, and to DNAs of T. gondii-positive anencephalic and hydrocephalic fetuses. It did not hybridize with DNA from controls, (i.e., normal human and baby pig peripheral blood leukocytes, spleen of normal mice, Plasmodium falciparum, Pneumocystis carinii, and pBR322). As few as 100 T. gondii parasites or 500 pg of purified DNA from T. gondii can be detected by dot-blot hybridization. This probe method was specific and sensitive, and has been used successfully in detecting various clinical cases of toxoplasmosis with T. gondii.

Animals↗
PubMed1992

Intragenic homozygous deletion of the WT1 gene in Wilms' tumor.

One example of intragenic homozygous deletion of the WT1 gene on chromosome 11p13 was found after screening 42 samples of Wilms' tumor DNA from Japanese patients. After construction of a restriction map for the genomic sequence covering the 3' half of the gene, the deletion was analysed at the nucleotide sequence level. The deletion occurred in the patient's germline on his paternal chromosome, and most of the short arm of his maternal chromosome 11 was subsequently lost in the tumor. The size of the deletion was about 8 kb, removing exons 6 and 7 and resulting in premature termination. The deletion seemed to be created by recombination between short homologous sequences found in an Alu repeat, with a 16-bp duplication left at the junction. This case conforms to a two-hit model for the genesis of a certain group of tumors, and supports the hypothesis that WT1 is one of the recessive oncogenes responsible for Wilms' tumor.

Base Sequence↗
PubMed1999

Physical mapping of a 670-kb region of chromosomes XVI and XVII from the human protozoan parasite Trypanosoma cruzi encompassing the genes for two immunodominant antigens.

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

Animals↗
PubMed1989

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject

Amino Acid Sequence↗
PubMed1997

The soybean cultivar specificity gene nolX is present, expressed in a nodD-dependent manner, and of symbiotic significance in cultivar-nonspecific strains of Rhizobium (Sinorhizobium) fredii.

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed nolXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to nolXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to nolXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2-4 kb DNA sequence that corresponds to nolX and its promoter region. Expression of nolX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. nolX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of nolX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. nolX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.

Bacterial Proteins↗
PubMed1994

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis.

NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation. Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.

Amino Acid Sequence↗
PubMed1994

Identification of the feline herpesvirus type 1 (FHV-1) genes encoding glycoproteins G, D, I and E: expression of FHV-1 glycoprotein D in vaccinia and raccoon poxviruses.

The genome of feline herpesvirus type 1 (FHV-1), the major cause of viral upper respiratory disease in cats, contains several genes encoding homologues of herpes simplex virus type 1 (HSV-1) glycoproteins. Restriction mapping studies have indicated that the group D genome of FHV-1 contains a unique short region that is 9.0 kb long. The nucleotide sequence of a 6.2 kb portion of this region was determined. Analyses of this sequence have identified five open reading frames capable of encoding homologues to HSV-1 protein kinase and glycoproteins gG, gD, gI and gE. Since gD of FHV-1 is most likely an immunologically important polypeptide, vaccinia and raccoon poxvirus recombinants expressing this glycoprotein were generated. In an indirect fluorescent antibody test these recombinants reacted strongly with a rabbit anti-FHV-1 serum. High titres of virus-neutralizing antibodies were also generated in rabbits inoculated with the vaccinia virus recombinant. A 53K viral polypeptide (gD) was detected with this antiserum on Western blots containing polypeptides from potassium tartrate-purified virions.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli.

A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed beta-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.

Animals↗
PubMed1996

The attenuated V60 strain of channel catfish virus possesses a deletion in ORF50 coding for a potentially secreted glycoprotein.

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion (approximately 1200 bp) in ORF50 of the V60 strain. Cloning and sequencing of part of this ORF confirmed the presence of a 1164-bp deletion. It should result in a protein of 282 amino acids instead of 670. The predicted truncated protein lacks most of a threonine-rich, highly repetitive region in its central part. Since the protein encoded by ORF50 possesses a hydrophobic N-terminal leader sequence and no membrane anchor sequence, we suggest that it could be a secreted glycoprotein. This protein might be N-glycosylated (35 potential sites) and, given the repetitive arrangement of its residues (mainly threonines), also heavily O-glycosylated like the mucin-type glycoproteins. The deletion observed in ORF50 of the V60 strain implies the loss of 24 potential N-glycosylation sites and should considerably reduce the extent of O-glycosylation.

Amino Acid Sequence↗