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Biomedical subjects

S Muthukrishnan

Publications and source records attributed to S Muthukrishnan.

At least 37 records · Page 2Linked to original sources
PubMed1993

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta.

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Amino Acid Sequence↗
PubMed1992

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice.

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Amino Acid Sequence↗
PubMed1992

Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.

Animals↗
PubMed1992

Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition.

Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium.

Journal Article↗
PubMed1991

Barley aleurone layer cell protoplasts as a transient expression system.

Protoplasts were prepared from barley aleurone layers using 'Onozuka' cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of alpha-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley alpha-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormone-responsive elements in hydrolase genes.

Abscisic Acid↗
PubMed1986

Study of barley endonucleases and alpha-amylase genes.

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.

Abscisic Acid↗
PubMed1984

Structural genes for alpha-amylases are located on barley chromosomes 1 and 6.

Genomic DNA from six euplasmic wheat-barley addition lines and from the wheat and barley parents were analyzed for the presence of alpha-amylase gene sequences, using plasmid DNAs from two different alpha-amylase cDNA clones as hybridization probes. Barley-specific alpha-amylase gene sequences were detected only in addition lines 1 and 6 carrying barley chromosomes 1 and 6, respectively. One cDNA clone probe hybridized strongly to sequences on chromosome 6 but not to those on chromosome 1. The other cDNA clone probe preferentially hybridized to sequences on chromosome 1. It is concluded that structural genes for alpha-amylases are located on barley chromosomes 1 and 6. alpha-Amylase genes on the same chromosome appear to be closely related to one another but substantially different from those on the other chromosome.

Chromosome Mapping↗
PubMed1984

Expression and regulation of alpha-amylase gene family in barley aleurones.

Several alpha-amylase clones were identified by screening a group of 345 cDNA clones derived from poly(A)+RNA from gibberellic acid (GA)-treated barley aleurone layer cells using an alpha-amylase cDNA clone that we had characterized previously as the hybridization probe. The alpha-amylase clones showed differences in the distribution of restriction enzyme sites, in the extent of cross hybridization, and in nucleotide sequence. There are at least three types of alpha-amylase clones in our collection, and the alpha-amylase cDNA clone reported by Rogers and Milliman [J. Biol. Chem. (1983) 258: 8169-8174] is a fourth type. The accumulation of alpha-amylase mRNAs in aleurones as a function of time after GA addition and of hormone concentration in the incubation medium was studied using different alpha-amylase cDNA clone probes. These studies indicate a differential control of the expression of the alpha-amylase genes by GA.

Base Sequence↗
PubMed1983

Hormonal control of alpha-amylase gene expression in barley. Studies using a cloned CDNA probe.

Starting with an enriched alpha-amylase mRNA preparation, cDNA was synthesized by reverse transcription and was cloned at the Pst I site of plasmid vector pBR322 by the "G-C tailing" procedure. Clones containing alpha-amylase cDNA sequences were identified among the recombinant clones by in vitro translation of hybrid-selected mRNA followed by immunoprecipitation of translation products with antiserum to alpha-amylase. The longest alpha-amylase cDNA clone isolated was 630 base pairs long and was characterized by digestion with restriction enzymes. Using this clone as a labeled nucleic acid hybridization probe, we observed that multiple bands of DNA with alpha-amylase sequences were present in restriction digests of barley embryo DNA. This indicates the presence of a family of barley alpha-amylase or alpha-amylase-like genes. Analysis of RNA blots with the cloned alpha-amylase hybridization probe indicated that alpha-amylase mRNA is synthesized de novo in barley aleurones after addition of gibberellic acid. The gibberellic acid-induced accumulation of alpha-amylase mRNA is blocked by cycloheximide which suggests the requirement of a newly synthesized protein factor for efficient expression of alpha-amylase gene.

Amylases↗
PubMed1979

Hormone-induced increase in levels of functional mRNA and alpha-amylase mRNA in barley aleurones.

Incubation of barley aleurone cells with gibberellic acid produces a progressive increase in the RNA content of the cells. The activity of poly(A)-containing RNA, measured in an in vitro wheat germ protein-synthesizing system, reaches a maximum approximately 12 hr after hormone addition and declines thereafter. The structurally intact functional mRNA content in these cells, measured as poly(A)-RNA with 5' "caps," also shows a maximum at 12 hr and correlates with the translational capacity of poly(A)-RNA. Activation of mRNA by guanylylation or methylation after addition of gibberellic acid is ruled out. Available evidence indicates that gibberellic acid stimulates protein synthesis by increasing the synthesis of mRNA. Studies with cycloheximide suggest that the induction of synthesis of alpha-amylase mRNA by gibberellic acid requires protein synthesis after hormone addition.

Journal Article↗
PubMed1978

5'-Terminal 7-methylguanosine and mRNA function. The effect of enzymatic decapping and of cap analogs on translation of tobacco-mosaic-virus RNA and globin mRNA in vitro.

1. Decapped tobacco mosaic virus (TMV) RNA and rabbit globin mRNA were prepared by enzymic treatment of RNAs with nucleotide pyrophosphatase purified from potato. The extent of removal of 5'-terminal 7-methylguanosine 5'-monophosphate (m7GMP) from TMV RNA was at least 97% as estimated by labeling of the 5' termini in vitro with S-adenosyl[methyl-3H]methionine catalysed by vaccinia virus methyltransferases. 2. The effect of enzymic decapping was compared with the effect of cap analogs on mRNAs translation in a nuclease-treated rabbit reticulocyte lysate and in a wheat germ extract. When translation was studied at low K+ concentration, little or no dependence on 5'-terminal 7-methylguanosine was found with either cell-free system. The importance of the 5'-terminal cap for the efficient translation of TMV RNA and globin mRNA increased as the concentration of K+ in a protein-synthesis system was raised. In a reticulocyte lysate analogs and enzymic decapping had a similar effect on translation. In a wheat germ extract, mRNA decapping resulted in a more pronounced decrease of mRNA activity, presumably due to the increased susceptibility of decapped mRNAs to the nucleases present in this protein synthesis system. 3. The requirement for a 5'-terminal cap was similar for the synthesis of 130,000-Mr and 165,000-Mr polypeptides coded by TMV RNA. This indicates that both proteins may be initiated at the common site close to the 5' terminus.

Animals↗
PubMed1978

Effect of methylation of the N6 position of the penultimate adenosine of capped mRNA on ribosome binding.

An RNA(2'-O-methyladenosine-N6)-methyltransferase isolated from HeLa cells was used to convert the ends of vaccinia virus mRNAs containing m7G(5')pppAm-to m7G(5')pppm6Am-. Under competitive conditions, there was no preferential binding of mRNAs containing m6Am residues within the capped ends relative to those containing Am either to wheat germ ribosomes at 70 mM potassium acetate or to reticulocyte ribosomes at 120 mM potassium acetate. Only at high concentrations of potassium acetate (200 mM) and at near-saturating mRNA concentrations, was a slight enrichment of mRNAs containing m6Am over those containing Am detected on reticulocyte ribosomes. It appears that unlike methylation of the N7 position of the terminal guanosine, methylation at the N6 position of the penultimate adenosine of mRNA has, at most, a small effect on binding to ribosomes under present in vitro conditions.

Adenosine↗
PubMed1978

The effect of "cap" analogs on reovirus mRNA binding to wheat germ ribosomes. Evidence for enhancement of ribosomal binding via a preferred cap conformation.

A variety of compounds related to the 5'-terminal "cap" (m7GpppN) of eukaryotic mRNA's were chemically synthesized and tested as inhibitors of reovirus mRNA binding to wheat germ ribosomes. Under our conditions of mRNA binding to ribosomes, 7-methyl-, 7-ethyl-, and 7-benzyl-GDP, but not GDP, decreased stable initiation complex formation by 70 to 80% at a concentration of 0.1 mM indicating that 7-substitution, but not a specific substituent, was required for the effect. Elimination of the positive charge on the imidazole of the 7-substituted compounds by treatment with alkali destroyed their inhibitory activity. Similarly, reduction to 8-hydro-m7GDP reversibly decreased the activity of m7GDP. The results were consistent with the hypothesis that the positive charge resulting from 7-alkylation provides an active cap conformer for binding via interaction of phosphate oxygens with the positively charged imidazole moiety. In accord with this suggestion, 7-carboxymethyl GDP and 7,8-dimethyl GDP were found to be less inhibitory than m7GDP. A 2-amino group was also important since m7IDP was less effective than m7GDP and 0.1 mM m7XDP did not inhibit ribosome binding. Other poor inhibitors were 6-Cl-m7GDP and 1,7-dimethyl GDP but N2,7-dimethyl GDP, 2'-deoxy-m7GDP, and m7GppI had essentially the same activity as m7GDP.

Guanosine Triphosphate↗
PubMed1978

Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA.

The ability of methylated vaccinia virus mRNA to bind to ribosomes derived from wheat germ of rabbit reticulocyte lysates has been studied after beta elimination, to remove the 5'-terminal m7G, and after "recapping" of beta-eliminated mRNA molecules using guanylyltransferase.guanine-7-methyltransferase complex from vaccinia virions. Removal of m7G from the mRNA results in significant loss of ability to bind to ribosomes and to simulate protein synthesis in vitro. Readdition of m7G, but not of unmethylated guanosine to the 5' end results in recovery of both of these functions. To evaluate the role of 2'-O-methylation of the penultimate ribonucleoside, mRNAs containing m7G-(5')pppA- and m7G(5')pppG- as well as m7G(5')pppAm- and m7G(5')pppGm- ends were synthesized in vitro at limiting S-adenosylmethionine concentrations by vaccinia virus cores. By comparing the cap sequences of ribosome-bound and unbound mRNAs, we concluded that 2'-O-methylation has at most a minor effect compared to that of m7G upon ribosome binding under in vitro conditions. Only at high input mRNA concentrations, at which competition might occur, was there some ribodomal enrichment of mRNAs containing a specific terminal structure, namely m7G(5')pppAm-.

Animals↗
PubMed1976

Influence of 5'-terminal m7G and 2'--O-methylated residues on messenger ribonucleic acid binding to ribosomes.

Removal of 80% of the 5'-terminal 7-methyl-guanosine (m7G) from methylated reovirus mRNA by beta elimination results in a concomitant loss of the ability to bind to wheat germ ribosomes. The mRNA molecules that retain the m7G account for most of the residual binding. Removal of the m7G from all molecules in preparations of methylated reovirus and vesicular stomatitis virus mRNA reduces the extent of binding to wheat germ ribosomes from 80% to 5-7%. In the reticulocyte lysate, however, significant binding (17-34%) of the beta-eliminated viral RNAs occurs. This m7G-independent binding appears to be due to recognition by ribosomes of other structural features of the 5'-proximal sequences. Initiation complexes involving beta-eliminated animal virus mRNAs and rabbit reticulocyte ribosomes appear to be more stable than the more heterologous combination of the same viral mRNAs and wheat germ ribosomes. In addition, evidence is presented to show that the 2'-O-methylated nucleoside of the 5'-terminal cap has a positive influence on the ribosome binding of viral mRNA and of capped synthetic ribopolymers. A model involving recognition of multiple structural features of the 5'-terminal region of mRNA by ribosomes during initiation of protein synthesis is presented.

Animals↗