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Biomedical subjects

B F Matthews

Publications and source records attributed to B F Matthews.

At least 19 recordsLinked to original sources
PubMed2003

Profiling local gene expression changes associated with Eimeria maxima and Eimeria acervulina using cDNA microarray.

Eimeria parasites show preferential sites of invasion in the avian intestine and produce a species-specific host immune response. Two economically important species, Eimeria acervulina and Eimeria maxima, preferentially invade and develop in the avian duodenum and jejunum/ileum, respectively. To investigate local host immune responses induced by parasite infection, global transcriptional changes in intestinal intraepithelial lymphocytes (IELs) induced by oral inoculation of chickens with E. acervulina or E. maxima were monitored using cDNA microarrays containing 400 unique chicken genes. Multiple gene transcripts were significantly up- or down-regulated following primary or secondary infection with E. acervulina or E. maxima. In general, infection by either parasite resulted in the expression changes of more genes following primary infection than following secondary infection, and E. acervulina caused more changes than did E. maxima. Although different regions of the small intestine were infected, similar changes in the levels of several cytokine mRNAs were observed in both Eimeria species following primary infection. Also identified was a set of transcripts whose expression was commonly enhanced or repressed in intestinal IELs of chickens infected with either parasite. Microarray analysis of chicken genes induced or repressed following Eimeria infection offers a powerful tool to enhance our understanding of host-parasite interactions leading to protective immunity.

Animals↗
PubMed2001

Physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene Rhg4.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.

Animals↗
PubMed2000

Detection of plant genes using a rapid, nonorganic DNA purification method.

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.

Base Sequence↗
PubMed1998

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase.

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.

Amino Acid Sequence↗
PubMed1997

Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase.

In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene. We previously cloned the soybean (Glycine max cv. Century) DapA gene in Escherichia coli to express functional soybean DS protein. Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine. Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications. When expressed in E. coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM.

Dose-Response Relationship, Drug↗
PubMed1997

Molecular cloning and expression of two cDNAs encoding asparagine synthetase in soybean.

Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparagine synthetase (AS; EC 6.3.5.4) were isolated. Their DNA sequences were determined and compared. The amino-terminal residues of the predicted SAS1 and SAS2 proteins were identical to those of the glutamine binding domain of AS from pea, asparagus, Arabidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS. The open reading frames of SAS1 and SAS2 encode a protein of 579 and 581 amino acids with predicted molecular weights of 65182 and 65608 Da respectively. Similarity of the deduced amino acid sequences of SAS1 and SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Arabidopsis; 70.5% and 72.5% for E. coli asnB and 61% and 63% for man. A plasmid, pSAS2E, was constructed to express the soybean AS protein in Escherichia coli. Complementation experiments revealed that the soybean AS protein was functional in E. coli. Southern blot analysis indicated that the soybean AS is part of a small gene family. AS transcript was expressed in all tissues examined, but higher levels were seen in stem and root of light-grown tissue and leaves of dark-treated tissue.

Algorithms↗
PubMed1995

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4.

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana.

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)(+)-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses. The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5' non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5' sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3' sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site. This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase.

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA, as a lacZ fusion protein was selected in a dapA- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.

Amino Acid Sequence↗
PubMed1994

Molecular genetics of the maize (Zea mays L.) aspartate kinase-homoserine dehydrogenase gene family.

Aspartate kinase (AK) and homoserine dehydrogenase (HSDH) are enzymes in the aspartate-derived amino acid biosynthetic pathway. Recent biochemical evidence indicates that an AK-HSDH bifunctional enzyme exists in maize (Zea mays L.). In this report, we characterize three genes that encode subunits of AK-HSDH. Two cDNAs, pAKHSDH1 and pAKHSDH2, containing the full-coding sequence, and one partial cDNA, pAKHSDH3, encode amino acid sequences similar to the reported monofunctional AK and HSDH enzymes from prokaryotes and yeast (Saccharomyces cerevisiae) and to AK-HSDH bifunctional enzymes of prokaryotes, yeast, carrot (Daucus carota), and Arabidopsis thaliana. Immunological and biochemical analyses verify that the cDNAs encode AK-HSDH and indicate that both the AK and HSDH activities are feedback inhibited by threonine. RNA blots identify a 3.2-kb transcript in all maize tissues examined. pAKHSDH1 and pAKHSDH2 map to chromosomes 4L and 2S, respectively. This study shows that maize contains AK-HSDH bifunctional enzyme(s) encoded by a small gene family of at least three genes. Maize AK-HSDH has conserved sequences found in communication modules of prokaryotic two-component regulatory systems, which has led us to propose that maize AK-HSDH may be involved in a similar regulatory mechanism.

Amino Acid Sequence↗
PubMed1993

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase.

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase.

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50,581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide. A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.

Amino Acid Sequence↗
PubMed1993

Cryptocotyle lingua in mullet, Chelon labrosus; significance of metacercarial excretory proteins in the stimulation of the immune response.

'O' group mullet, Chelon labrosus, were experimentally infected with Cryptocotyle lingua (Heterophyidae) by tail dip in a suspension of cercariae. Metacercariae were excised after 1 and 24 hours and prepared for TEM and post-embedding immunogold labelling. Antisera to cercariae of C. lingua were raised in adult mullet by natural infection via the skin and by intra-peritoneal injection of sonicate. The membrane-bound vesicles within the syncytial lining of the metacercarial excretory vesicle were found to be intensely antigenic with both antisera; the epidermal secretory bodies type 5 within the cystogenous glands gave a positive response. Penetration gland contents were not found to be antigenic with either antiserum. Discharge of the membrane-bound vesicles coinciding with both the reorganization of the lining of the metacercarial excretory vesicle and with cyst wall formation appears to be of significance in the initiation of the host immune response. That the term 'excretory vesicle' in Digenea may be a misnomer is discussed in the light of current information regarding the wide range of functions attributed to this structure.

Animals↗
PubMed1992

Identification and expression of a cDNA clone encoding aspartate aminotransferase in carrot.

A full-length cDNA clone encoding aspartate aminotransferase (AAT) has been identified from a carrot root cDNA library. Degenerate oligo primers were synthesized from the known amino acid sequence of AAT form I from carrot (Daucus carota L. cv Danvers). These primers were utilized in a polymerase chain reaction to amplify a portion of a carrot AAT gene from first strand cDNA synthesized from poly(A)(+) RNA isolated from 5-d-old cell suspension cultures. The resulting 750-bp fragment was cloned, mapped, and sequenced. The cloned fragment, mpAAT1, was used as a probe to identify a full-length cDNA clone in a library constructed from poly(A)(+) RNA isolated from carrot roots. A 1.52-kb full-length clone, AAT7, was isolated and sequenced. AAT7 has 54% nucleotide identity with both the mouse cytoplasmic and mitochondrial AAT genes. The deduced amino acid sequence has 52 and 53% identity with the deduced amino acid sequences of mouse cytoplasmic and mitochondrial AAT genes, respectively. Further analysis of the sequence data suggests that AAT7 encodes a cytoplasmic form of carrot AAT; the evidence includes the (a) absence of a transit or signal sequence, (b) lack of "m-residues," or invariant mitochondrial residues, in the carrot AAT sequence, and (c) high degree of sequence similarity with the amino acid sequence previously obtained for form I of carrot, a cytoplasmic isoenzyme. High- and low-stringency hybridizations to Southern blots of carrot nuclear DNA with AAT7 show that AAT is part of a small multigene family. Northern blot analysis of AAT7 suggests that AAT is expressed throughout cell culture up to 7 d and is highly expressed in roots but not in leaves.

Journal Article↗
PubMed1991

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs.

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.

Amino Acid Sequence↗
PubMed1991

Lecithochirium furcolabiatum (Jones 1933), Dawes 1947: the miracidium and mother sporocyst.

Experimental infections of the marine topshell Gibbula umbilicalis with Lecithochirium furcolabiatum (Digenea: Hemiuroidea) have allowed the development of a model system which will enable further studies of the molluscan host response. The long-lived intertidal prosobranch host is easily maintained in the laboratory, and experimental infection rates of 98% were consistently achieved. The miracidium and mother sporocyst have been studied at both light and ultrastructural levels, providing the first account of the morphology of these stages in Hemiuridae. The ingested egg hatches within the host intestine, treatment with L-cysteine and alkaline pH stimulating miracidial emergence in vitro. The general body surface of the miracidium is devoid of spines or cilia, the latter being restricted to four plates near the anterior extremity. The miracidium swims actively prior to penetration of the gut wall, the sporocyst being released from the miracidial epidermal coat within the haemocoel. Within 5 weeks of infection, the filamentous mother sporocyst contains 1 to 3 oval germ balls, daughter sporocysts being recorded free within the digestive gland haemocoel 7 weeks later. Twenty three weeks after ingestion of eggs, the daughter sporocyst extends into the host gill filaments, containing cystophorous cercariae ready for emergence.

Animals↗