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PubMed1996

Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart.

We previously demonstrated that conduction-system myocytes of the rabbit heart express cytoskeletal proteins immunologically related to neurofilaments. In order to determine more precisely the nature of these proteins, we screened an expression cDNA library, prepared from the sino-atrial node region of the rabbit heart, using a monoclonal antibody which reacts with the M subunit of neurofilaments. Sequence analysis of the isolated cDNA clones shows high homology with rat and human neurofilament M mRNAs. Northern blot analysis demonstrates hybridization with a transcript expressed in brain, with the size expected for neurofilament M mRNA. An mRNA species of the same size is also detectable in the Northern blot of the sino-atrial node region RNA. In situ hybridization documents that in the adult rabbit the transcript accumulates in neurons and is localized in myocytes of the sino-atrial and atrio-ventricular nodes and of the atrio-ventricular bundle and bundle branches, but not in working atrial and ventricular myocytes. Developmental analysis was undertaken in order to determine the distribution of the neurofilament M mRNA in the rabbit embryonic heart. In situ hybridization shows that neurofilament M mRNA is detectable in a few ventricular myocytes in proximity to the atrio-ventricular groove after 9.5 days of embryonic development and it is accompanied by the presence of the protein. At subsequent stages of development neurofilament M mRNA is detectable in a number of cardiac myocytes, which are mainly localized at the atrio-ventricular junction and in the ventricular subendocardium and presumably correspond to myocytes of the heart conduction system.

Amino Acid Sequence↗

PubMed1996

Cloning Expressed cDNAS from Defined Chromosomal Regions Using Interspecific Microcell Hybrids and Subtractive Hybridization

Genes expressed from a defined genomic interval can be cloned using interspecific microcell hybrids and subtractive hybridization. This approach involves identifying a pair of interspecific hybrids that contain nearly isogenic fragments of a relevant chromosome, except for a region of nonoverlap encompassing the locus of interest. Radiolabeled cDNAs are synthesized from the hybrid cell line that contains the region of interest and hybridized in solution with an excess of biotinylated mRNA from the microcell hybrid lacking the region of interest. Subtracted cDNA probes enriched for sequences expressed from the nonoverlap region are isolated by the addition of streptavidin followed by organic extraction. These subtracted cDNA probes are used to screen a cDNA library prepared from an appropriate tissue, and candidate cDNAs are mapped by Southern hybridization to determine whether they are encoded within the nonoverlap region. This technique is an attractive alternative to other methods for identifying genes in a genomic region because it does not require that the gene of interest be localized to a minimal genetic interval, and the genomic DNA encompassing the region need not be cloned. Additionally, the method detects only expressed genes, simplifying the search for candidate genes within a genomic region.

Journal Article↗

PubMed2000

An RNA helicase, RHIV -1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13.

The impact of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine alveolar macrophages (Mo) was examined by differential display reverse transcription PCR (DDRT-PCR). A PRRSV-induced expressed gene tag (EST) was used to isolate and identify a single cDNA clone from a library prepared from porcine peripheral blood. Rapid amplification of cDNA ends (RACE) was employed to clone a 1.5 kb fragment at the 5' end of the mRNA. DNA sequencing identified an open reading frame (ORF) of 2820 bp. Deduced amino acid sequence revealed the eight conserved domains characteristic of the DEAD/H box protein superfamily. The putative porcine RNA helicase induced by virus (RHIV -1) showed 84% amino acid similarity to human retinoic acid-induced gene (RIG-I). Porcine RHIV -1 transcripts were ubiquitously expressed in various pig tissues, while in PRRSV-infected pigs, higher expression was observed in several tissues persistent for PRRSV. These data indicate the association of PRRSV genome replication with enhaced host cell RNA helicase gene expression. Finally, the RHIV -1 gene was localized on porcine chromosome 10q13 between markers SSC25A02 and SWR334 via somatic cell panel and radiation hybrid (RH) mapping strategies.

Amino Acid Sequence↗

PubMed1993

Class I major histocompatibility complex genes of the red-necked Wallaby, Macropus rufogriseus.

Marsupials are one of three main evolutionary lineages in mammals, the other two being the monotremes and the placental mammals. The marsupial and the placental lineages separated between 120 and 156 million years ago. In this communication, we provide the first molecular description of class I major histocompatibility complex (Mhc) genes in a representative of the marsupial lineage, the red-necked wallaby, Macropus rufogriseus. Three different, nearly full-length class I Mhc sequences were identified in the cDNA library prepared from spleen mRNA of a single wallaby. The three sequences identify at least two loci. Under the assumption that two of the identified sequences are alleles, we designate the three wallaby genes Maru-Mhc-UA*01, Maru-Mhc-UA*02, and Maru-Mhc-UB*01. The three Maru sequences share several codon deletions and insertions not found in the class I genes of placental mammals. Comparisons of genetic distances among the known class I genes suggest that the Maru genes arose from one ancestral element, whereas the class I genes of the placental mammals arose from another, different ancestral element. The absence of an identifiable defect in the three Maru sequences suggests that the genes from which they were derived are functional. Hence, as in placental mammals, there appear to be two functional class I Mhc loci in the marsupials as well.

Animals↗

PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗

PubMed1995

Molecular cloning, characterization and expression analysis of two catalase isozyme genes in barley.

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and -3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).

Amino Acid Sequence↗

PubMed1993

Molecular characterization of a gene encoding a cysteine-rich protein preferentially expressed in anthers of Lycopersicon esculentum.

The tomato (Lycopersicon esculentum Mill.) cDNA clone TomA5B was isolated by differential screening of a cDNA library prepared from anthers at late meiosis to tetrad formation. The 5B gene is present in a single copy in the tomato genome. Expression is developmentally regulated and tissue specific. RNA accumulation was detected from premeiosis through tetrad release in the tapetal cell layer of the anther with low levels of RNA detected in petals and early stages of pistil development. The protein deduced from the DNA sequence analysis is predicted to have a molecular mass of 11.1 kDa and a secretory signal sequence, suggesting it is a secreted protein. The deduced 5B protein has a pattern of cysteine residues that is similar to other proteins that have stamen-specific expression and to a superfamily of seed proteins. The 5B protein is unique in that there is no amino acid sequence similarity to other proteins beyond the similar cysteine motif.

Amino Acid Sequence↗

PubMed1994

A putative O-methyltransferase from barley is induced by fungal pathogens and UV light.

A cDNA clone, pBH72-F1 (F1), was isolated from a cDNA library prepared from barley leaves 72 h after inoculation with Erysiphe graminis f.sp. hordei. The 1388 bp nucleotide sequence of pBH72-F1 contains an open reading frame encoding a 42.3 kDa polypeptide of 390 amino acids which shows sequence similarity to O-methyltransferases (OMTs) from different plant species; the highest identity (41%) was observed with a putative OMT expressed in roots of maize. A phylogenetic analysis shows that the barley and maize sequences are distinctly different from the ortho-diphenol-OMTs involved in lignin formation. A putative S-adenosyl-L-methionine-binding motif (KELVDDSITN) determined for a rabbit protein-carboxyl OMT is partially conserved in the encoded amino acid sequence. Genomic Southern blot hybridization shows that pBH72-F1 probably represents a single copy gene. The F1 clone corresponds to a gene transcript exhibiting a relatively late accumulation in mildew-infected barley leaves compared to other pathogen-induced transcripts, such as transcripts encoding PR proteins, a peroxidase, and transcripts homologous to a maize caffeic acid OMT. No transcript was detected in plants exhibiting papilla resistance at time points when resistance is thought to be manifested. The atypical transcript accumulation pattern for F1 was also observed after infection by other pathogens and after UV-light treatment.

Amino Acid Sequence↗

PubMed1994

Biphasic and differential expression of cytosolic glutamine synthetase genes of radish during seed germination and senescence of cotyledons.

Three structurally distinct cDNA clones for cytosolic glutamine synthetase (GS1) were isolated from libraries prepared from senescing radish cotyledons. Northern blot analysis showed that transcripts from two of the three genes encoding GS1, Gln1;1 and Gln1;3, accumulated in the cotyledons during both dark-induced and natural senescence. Transcripts from the last gene, Gln1;2, remained at a low level during both processes. Transcripts from all three Gln1 genes accumulated in cotyledons of germinating seeds. We infer from these findings that GS1 enzymes function in both germination and senescence to convert ammonium to glutamine to remobilize nitrogen from source to sink organs. We have also examined the pattern of expression of these genes in different tissues. All three genes are expressed in roots. A large amount of transcripts from Gln1;1 accumulated in hypocotyls. Whereas none were transcribed in flowers. During dark-induced senescence of cotyledons, application of inorganic nitrogen delayed chlorophyll degradation. Inorganic nitrogen enhanced the accumulation of Gln1;1 transcripts, but decreased those of Gln1;3. In contrast, application of glutamine promoted yellowing of cotyledons during the dark treatment, and slightly increased the amounts of transcripts from Gln1;3 but decreased those of Gln1;1. Transcription of the three Gln1 genes appears, therefore, to be differentially regulated in radish cotyledons during senescence and germination.

Amino Acid Sequence↗

PubMed1994

Characterization of four new beta-tubulin genes and their expression during male flower development in maize (Zea mays L.).

Four different beta-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of alpha-tubulin genes described previously. Hybridization probes from the 3' non-coding regions of six beta-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the beta-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

Amino Acid Sequence↗

PubMed1995

Molecular characterization of a glyoxysomal citrate synthase that is synthesized as a precursor of higher molecular mass in pumpkin.

A cDNA clone for glyoxysomal citrate synthase (gCS) was isolated from a lambda gt11 cDNA library prepared from etiolated pumpkin cotyledons. The cDNA of 1989 bp consisted of a 1548 bp open reading frame that encoded 516 amino acid residues. The deduced amino acid sequence of gCS did not have a typical peroxisomal targeting signal at its carboxyl terminal. A study of expression in vitro of the cDNA and an analysis of the amino-terminal sequence of the citrate synthase indicated that gCS is synthesized as a larger precursor that has a cleavable amino-terminal presequence of 43 amino acids. The predicted amino-terminal sequence of pumpkin gCS was highly homologous to those of other microbody enzymes, such as 3-ketoacyl-CoA thiolase of rat and malate dehydrogenase of watermelon that are also synthesized as precursors of higher molecular mass. Immunoblot analysis showed that the level of gCS protein increased markedly during germination and decreased rapidly during the light-induced transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the level of mRNA for gCS reached a maximum earlier than that of the protein and declined even in darkness. The level of the mRNA was low during the microbody transition. These results indicate that the accumulation of the gCS protein does not correspond to that of the mRNA and that degradation of gCS is induced during the microbody transition, suggesting that post-transcriptional regulation plays an important role in the microbody transition.

Animals↗

PubMed1996

A low-temperature-responsive gene from barley encodes a protein with single-stranded nucleic acid-binding activity which is phosphorylated in vitro.

A low-temperature-responsive gene, blt 801, isolated from a winter barley (Hordeum vulgare L.) cDNA library prepared from leaf meristematic tissue, was sequenced. The deduced amino acid sequence predicts a glycine-rich RNA-binding protein (GR-RNP) which was homology to stress-responsive GR-RNPs from several other plant species. BLT 801 is a two-domain protein, the amino-terminal domain comprises a consensus RNA-binding domain similar to that found in many eukaryotic genes and the carboxy-terminal domain is extremely glycine-rich (68.5% glycine). Blt 801 mRNA also accumulates in response to the phytohormone abscisic acid. The protein encoded by blt 801 has been produced as a recombinant fusion protein using a bacterial expression vector. The fusion protein, a chimaera of glutathione S-transferase and BLT 801, has been used in studies to determine nucleic acid binding and other characteristics. Binding studies with single-stranded nucleic acids show that BLT 801 has affinity for homoribopolymers G, A and U but not C, it also binds to single-stranded DNA and selects RNA molecules containing open loop structures enriched in adenine but low in cytosine. Blt 801 has a consensus motif for phosphorylation by cAMP protein kinase (PKA) at the junction between the two domains which can be phosphorylated by PKA in vitro and which, by analogy to animal studies, may have significance for controlling enzyme function.

Amino Acid Sequence↗

PubMed1993

The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors.

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.

Amino Acid Sequence↗

PubMed1993

Molecular analysis and spatial expression pattern of a low-temperature-specific barley gene, blt101.

A cDNA clone of the previously unreported low-temperature-induced gene blt101 was isolated after a differential screen of a cDNA library prepared from low-temperature (6 degrees C day/2 degrees C night) grown barley shoot meristems. Southern blot analysis of barley ditelosomic addition lines was used to assign this single-copy gene to the long arm of chromosome 4. Analysis of steady-state levels of blt101 mRNA showed the induction of this transcript in shoot meristems upon transfer of barley (cv. Igri) plants from control (20 degrees C/15 degrees C) to low (6 degrees C/2 degrees C) temperature treatment. Further, the high level of this transcript is maintained at low temperatures but is reduced on transfer from low to control temperatures. The gene is not induced by drought or by foliar application of ABA. Analysis of segregating doubled haploid lines shows that there is no specific association of this gene with either spring/winter growth habit or frost hardiness. Examination of the spatial expression pattern revealed ubiquitous expression of blt101 in low-temperature (6 degrees C/2 degrees C) grown barley shoot meristems, mature leaves and roots.

Abscisic Acid↗

PubMed1992

Iron induces ferritin synthesis in maize plantlets.

The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH2-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 microM Fe-EDTA/75 microM Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)+ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5' extremity, were characterized. They are identical, except in their 3' untranslated region: FM1A extends 162 nucleotides beyond the 3' terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5' untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

Amino Acid Sequence↗

PubMed1989

Purification of (1-->3)-beta-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone.

A (1-->3)-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) of apparent M(r) 32,000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (1-->3)-beta-glucanases GI and GII have pI values of 8.6 and > or = 10.0, respectively. Comparison of the sequences of their 40 NH2-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (1-->3)-beta-glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (1-->3)-beta-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1-->3)-beta-glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (1-->3)-beta-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1-->3, 1-->4)-beta-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1-->3)-beta-glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (1-->3, 1-->4)-beta-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.

Amino Acid Sequence↗

PubMed1993

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors.

We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defence-induced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systematically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of (1-->3, 1-->4)-beta-glucan endohydrolases from germinated wheat (Triticum aestivum).

A (1-->3, 1-->4)-beta-glucan 4-glucanohydrolase [(1-->3, 1-->4)-beta-glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (1-->3, 1-->4)-beta-glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (1-->3, 1-->4)-beta-glucanase isoenzyme EI from barley. The complete primary structure of the wheat (1-->3, 1-->4)-beta-glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)+ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated lambda LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH2-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32,085 and a predicted pI of 8.1. The other cDNA, designated lambda LW1, carries a 109 nucleotide pair sequence at its 5' end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3'-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.

Amino Acid Sequence↗