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A Leite

Publications and source records attributed to A Leite.

31 records · Page 2Linked to original sources
PubMed1994

Structural characterization and promoter activity analysis of the gamma-kafirin gene from sorghum.

A genomic clone encoding the gamma-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of gamma-kafirin with the published sequences of gamma-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in gamma-zein, four times in gamma-kafirin and three times in gamma-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of gamma-prolamins. Several putative regulatory sequences common to the gamma-kafirin and gamma-zein genes were identified in both the 5' and the 3' flanking regions. Putative GCN4-like regulatory sequences were found at positions -192 and -476 in the 5' flanking region of gamma-kafirin. In the 3' noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions +658, +716, and +785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the gamma-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of beta-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.

Amino Acid Sequence↗
PubMed1994

The transcriptional activator Opaque2 recognizes two different target sequences in the 22-kD-like alpha-prolamin genes.

The maize Opaque2 (O2) protein is a "leucine zipper" DNA binding factor that interacts with the sequence TCCACGTAGA in the promoters of the 22-kD alpha-zein genes and activates its transcription. A completely different consensus sequence (GATGAPyPuTGPu) identified in b-32, a gene that encodes an abundant albumin that is also under control of the O2 locus, can also be bound by the O2 protein. We showed that the gene encoding the 22-kD-like alpha-coixin, the alpha-prolamin of the maize-related grass Coix, can also be transactivated by the O2 protein. A binding assay in vitro and footprint analysis demonstrated that the GACATGTC sequence of the alpha-coixin promoter can be recognized and protected by the maize O2 protein. Employing transient expression experiments in immature maize endosperm and tobacco mesophyll protoplasts, we demonstrated that the O2 protein can activate expression of the beta-glucuronidase reporter gene placed under the control of the 22-kD-like alpha-coixin promoter. We also demonstrated that a 22-kD-like alpha-coixin pseudogene promoter is transactivated by the maize O2 protein.

Base Sequence↗
PubMed1993

Studies of the zein-like alpha-prolamins based on an analysis of amino acid sequences: implications for their evolution and three-dimensional structure.

alpha-Prolamins are the major seed storage proteins of species of the grass tribe Andropogonea. They are unusually rich in glutamine, proline, alanine, and leucine residues and their sequences show a series of tandem repeats presumed to be the result of multiple intragenic duplication. Two new sequences of alpha-prolamin clones from Coix (pBCX25.12 and pBCX25.10) are compared with similar clones from maize and Sorghum in order to investigate evolutionary relationships between the repeat motifs and to propose a schematic model for their three-dimensional structure based on hydrophobic membrane-helix propensities and helical "wheels." A scheme is proposed for the most recent events in the evolution of the central part of the molecule (repeats 3 to 8) which involves two partial intragenic duplications and in which contemporary odd-numbered and even-numbered repeats arise from common ancestors, respectively. Each pair of repeats is proposed to form an antiparallel alpha-helical hairpin and that the helices of the molecule as a whole are arranged on a hexagonal net. The majority of helices show six faces of alternating hydrophobic and polar residues, which give rise to intersticial holes around each helix which alternate in chemical character. The model is consistent with proteins which contain different numbers of repeats, with oligomerization and with the dense packaging of alpha-prolamins within the protein body of the seed endosperm.

Amino Acid Sequence↗
PubMed1993

Sequence analysis of 22 kDa-like alpha-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements.

Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of a cDNA encoding a sulfur-rich coixin.

A full-length cDNA clone encoding a sulfur-rich Coix prolamin was isolated using a cDNA library constructed from polysomal mRNA prepared from immature Coix endosperm. The deduced amino acid sequence of the cDNA clone predicted a polypeptide of 194 residues, which shared 64% homology with the 17 kDa beta-zein. The mature protein contains the familiar composition of the prolamins and an unusually high content of the sulfur-containing amino acids methionine (11.6%) and cysteine (5.2%). In vitro transcription followed by in vitro translation of the coding region of the pBCX17.9S clone gave rise to a polypeptide with an apparent molecular weight corresponding to the C4 alpha-coixin. Hydropathy analysis showed that C4 alpha-coixin is slightly more hydrophobic than beta-zein.

Amino Acid Sequence↗
PubMed1992

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms.

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and alpha-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to alpha-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, alpha-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Journal Article↗
PubMed1990

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis.

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain alpha-, beta-, and gamma-zein and alpha-, beta-, and gamma-coixin. The alpha-coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa alpha-zeins. Like the alpha-zeins, the C1 and C2 alpha-coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to gamma-coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of gamma-zein and represents 15% of the total coixin. The beta-zein fraction was composed of a major 17 kDa protein band, while the beta-coixin fraction consisted of a mixture of alpha- and gamma-coixins. Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa alpha-zein, as did C4 and C5 antisera. The antiserum against gamma-coixin showed strong cross-reaction with gamma-zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa alpha-zeins as well as the 28 and 16 kDa gamma-zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa alpha-zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1989

Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.

Cellulose↗
PubMed1988

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle.

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.

Animals↗
PubMedDate not supplied

[Dystrophia myotonica].

The authors present two cases of myotonic dystrophy (Steinert's disease) with cardiac involvement in young adults. They point out the rareness of this disease, the multisystemic involvement and also the diagnostic problems in the absence of the classic clinical picture.

Adult↗