An unusual pattern of genome organization in two Phaseolus plant species.

The modes of DNA sequence organization in the nuclear genomes of French bean and green gram are studied. The reassociation kinetics of DNAs of increasing fragment lengths have revealed that approximately 60% of the genomes of French bean and green gram consist of interspersed repeated and single copy sequences. The hyperchromicity and S1-nuclease-resistance of the reassociated long DNA fragments have further confirmed the occurrence of interspersion and have shown the actual proportion of repetitive DNA to be 35-40% in each species. These experiments have also yielded the size of interspersed repeated sequences as 1900-2350 nucleotide pairs. The fragment length of the interspersed single copy DNA sequences is estimated from a curve relating the fraction of DNA fragments binding to hydroxyapatite and the DNA fragment length and is 1300 nucleotides in both plant species. Approximately 35-40% of the single copy sequences are interspersed in this manner in French bean and green gram. Since the lengths of the interspersed repeated sequences are significantly different from those in other plant species, the DNA sequence organization patterns in French bean and green gram are considered to be unusual.

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.

Analysis of plant genomes. III. Denaturation and reassociation properties of cryptic satellite DNAs in barley (Hordeum vulgare) and wheat (Triticum aestivum).

A cryptic satellite fraction was isolated from barley and wheat by preparatory ultracentrifugation of total DNA in Ag+-Cs2SO4 density gradients and was characterized by studying its denaturation-reassociation properties. Wheat satellite DNA underwent thermal denaturation as a single component with a Tm of 81 degrees C while barley satellite DNA consisted of one major (Tm = 82.5 degrees C) and one minor (Tm = 91 degrees C) component. When the barley and wheat satellites were reassociated and then melted, the Tm values were found to be 6--7 degrees C lower than those of the corresponding native DNA preparations. Examination of the C0t curves of these two satellite DNAs revealed the presence of a major, fast reassociating and a minor, slow reassociating fraction. The fast reassociating DNA fraction of barley was found to have a complexity of 9.7 . 10(5) daltons while that of wheat satellite was 5.8 . 10(5) daltons. Since these satellites reassociated with about 4--5% base mismatching, as judged by their deltsTm (6--7 degrees C), they each appear to consist of rather similar base sequences.

Effects of commonly used antidiabetic drugs on antioxidant enzymes and liver function test markers in type 2 diabetes mellitus subjects - pilot study.

OBJECTIVE: The present study analyzed the effects of antidiabetic drugs on antioxidant enzymes and liver function test (LFT) markers and their association with homeostatic model assessment of insulin resistance (HOMA-IR) in type 2 diabetic subjects. METHODS: We assessed healthy and diabetic subjects (100 each). Diabetic subjects were divided based on treatment with only metformin, metformin in combination with other antidiabetic drugs and insulin in combination with other antidiabetic drugs. LFT markers, antioxidant status and HOMA-IR were assessed in the subjects. RESULTS: Superoxide dismutase activity was higher (p<0.01) while catalase activity was lower (p<0.01) in the diabetic subjects as compared to controls. Serum glutamate-pyruvate transaminase (SGPT) (p<0.01) and bilirubin (p<0.05) levels were higher in diabetic male subjects while urea (p<0.05) levels were lower and SGPT (p<0.01) levels were higher in diabetic female subjects. In male subjects consuming only metformin, a positive association between HOMA-IR and insulin (p<0.05) was seen. A positive association between HOMA-IR and glucose (p<0.01), insulin (p<0.01), SOD (p<0.01) and SGPT (p<0.05) was seen in males receiving metformin with other drugs. Interestingly, the female subjects on metformin displayed a positive association between HOMA-IR and insulin (p<0.05) only. A positive association of HOMA-IR with glucose (p<0.01) and insulin (p<0.05) was seen in females on metformin in combination with other anti-diabetic drugs. CONCLUSION: The alterations in the antioxidant enzyme activities and liver function tests are dependent upon the gender and glycemic status of subjects while the variations in correlations of HOMA-IR with antioxidant enzymes, liver function tests and inflammatory markers are dependent on type of treatments.

Amino acid repeat patterns in protein sequences: their diversity and structural-functional implications.

All the protein sequences from SWISS-PROT database were analyzed for occurrence of single amino acid repeats, tandem oligo-peptide repeats, and periodically conserved amino acids. Single amino acid repeats of glutamine, serine, glutamic acid, glycine, and alanine seem to be tolerated to a considerable extent in many proteins. Tandem oligo-peptide repeats of different types with varying levels of conservation were detected in several proteins and found to be conspicuous, particularly in structural and cell surface proteins. It appears that repeated sequence patterns may be a mechanism that provides regular arrays of spatial and functional groups, useful for structural packing or for one to one interactions with target molecules. To facilitate further explorations, a database of Tandem Repeats in Protein Sequences (TRIPS) has been developed and is available at URL: http://www.ncl-india.org/trips.

Potential of (GATA)n microsatellites from rice for inter- and intra-specific variability studies.

BACKGROUND: The microsatellite, (GATA)n has been frequently used for DNA fingerprinting. However, very few attempts have been made to analyze (GATA)n-containing loci in rice. RESULTS: Three polymorphic (GATA)n-harboring loci viz. OS1A6, OS1H10 and OS2E7, containing 7-13 repeat motifs were identified from a genomic library of a cultivated rice, Oryza sativa var. Basmati-370 using oligonucleotide probe (GATA)4. When (GATA)n flanking primers were used to screen 26 wilds (representing different genomes of rice), 16 cultivars, 47 Indian elite rice varieties and 37 lines resistant/susceptible to bacterial blight, up to 22 alleles were obtained at an individual locus. Also, interestingly the bacterial blight resistant lines clustered into a separate group from the remaining rice genotypes, when a dendrogram was constructed based on the polymorphism obtained at the three loci. This may be due to the partial homology of the clones OS1H10 and OS2E7 to regions encoding O. longistaminata receptor kinase-like protein and pathogenesis-related protein. The ability of these O. sativa flanking primers to amplify DNA of maize, wheat, barley and oat indicates that these (GATA)n-containing loci are conserved across different cereal genera. CONCLUSIONS: The large allele number obtained reveals the potential of (GATA)n-containing loci as powerful tools to detect simple sequence length polymorphism (SSLP). The (GATA)n-flanking primers were not only useful in distinguishing between closely related genotypes, but could also be used for cross-species amplification and are also conserved across different cereal genera. These loci could also cluster the bacterial blight resistant/susceptible lines into different groups based on the resistance genes present in them.

Ty1-copia retrotransposon-like elements in chickpea genome: their identification, distribution and use for diversity analysis.

Ty1-copia retrotransposon-like elements were amplified from Cicer species using primers derived from the conserved region of the reverse transcriptase gene. Two fragments, of size approximately 280bp and approximately 650 bp, were obtained, which on sequencing showed homology for the Ty1-copia reverse transcriptase region. Interestingly, the approximately 650 bp fragment showed two reverse transcriptase regions, one from Ty1-copia and the other from Tto1 element fused together. The copy number was high in the cultivated Cicer arietinum genome compared with the wild Cicer reticulatum. Genetic diversity among the Cicer species was investigated using the conserved primers which grouped the wild species and the cultivated C. arietinum separately.

Cloning and characterization of the adenine phosphoribosyltransferase-encoding gene (APT1) from Saccharomyces cerevisiae.

We have cloned, sequenced and characterized the APT1 (adenine phosphoribosyltransferase) gene from Saccharomyces cerevisiae. The APT1 sequence includes an open reading frame encoding 221 amino acids and is contained within a 1322-bp insert that complements APRT-deficient mutants to wild-type levels of enzyme activity. Analysis by primer extension revealed multiple transcription start points (tsp) and a major tsp 21-bp upstream from the ATG start codon. A transcript initiated at the major tsp would yield a 700-nt mRNA which is in agreement with the size observed by Northern analysis. Sequence comparison indicates that the yeast enzyme shares strong similarities with other known APRT of bacterial, invertebrate, plant and mammalian origins.

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest.

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.

Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli.

A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases.

Biochemical and immunological characterization of rice albumin.

Rice albumin from Oryza sativa (Var. Basmati 370) accounts for about 5% of the total seed proteins. A major fraction of rice albumin has been found to be a glycoprotein which is a monomer of 60 kd having iso-electric point 6.54. When rice albumin is digested with trypsin, it shows the presence of 24 peptides as against 28 peptides which were estimated from its amino acid composition. This indicates the presence of a few peptides which resemble each other in their charge and Rf values. Antibodies against Con A purified rice albumin were affinity purified and were used to quantitate the rice albumin levels during post-anthesis by RIA and ELISA. The latter experiments reveal that maximum albumin is present between 18 and 20 days post-anthesis.

A sensitive and rapid immunochemical method for quantitation of proteins.

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.

Identification of a dispersed MboI repeat family in five higher plant genomes.

Digestion of nuclear DNAs of five plants, namely Cucurbita maxima (red gourd), Trichosanthes anguina (snake gourd), Cucumis sativus (cucumber), Cajanus cajan (pigeon pea) and Phaseolus vulgaris (french bean) with the restriction endonuclease MboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. The MboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts of MboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.

Novel application of quantitative immunoassays for screening seed globulins of cowpea varieties.

Using antibodies raised in rabbits, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are standardized for cowpea (var. Pusa Barsati) seed globulins. The RIA, when used to screen three stages of seed development, reveals that maximum globulins are detected at 28 days after flowering. When three different varieties of cowpea are assayed for their globulin content by RIA and ELISA, it is observed that the Bold Grain cowpea has the highest amount of related globulin as compared to two other varieties, namely Pusa Phalgun and Asparagus Bean.

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase.

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum.

The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.

Studies of operational variables in batch mode for genetically engineered Escherichia coli cells containing penicillin acylase.

A recombinant Escherichia coli was constructed by cloning the penicillin acylase gene from E. coli ATCC 11105. The cloning was carried out using a recombinant plasmid pUSAD2 harboring the pac gene. The recombinant E. coli DH 5 cells were used as a biocatalyst and were studied in a batch reactor for determination of optimum value for some of the process parameters, such as effect of pH, temperature, substrate concentration, kLa and effect of carbon and nitrogen source on penicillin acylase production. These values were then compared with the values obtained with the standard parent strain. Whereas the cloned pac gene was found to produce higher levels of penicillin acylase constitutively, the process parameters remained about the same for both the parent and the recombinant.

Use of high resolution thermal denaturation approach in preliminary identification of plant DNAs.

High resolution thermal denaturation (HRTD) profiles of the DNAs of six related millets, oat and rice showed several maxima and shoulders skewed towards the GC-rich side. Linear regression and correlation analyses exhibited no correlation between the repetitive DNA content/nuclear DNA content and the number of peaks, while there existed a very good correlation between delta T and repetitive DNA content/nuclear DNA content indicating that increase in repetitive DNA content has resulted in a greater sequence heterogeneity in these DNAs. In addition, specific melting characteristics of each of the eight plant DNAs were identified, which showed species specificity.