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.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary 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.NCL Communication No.: 4307     

Quantitation of rice glutelin,an acid/alkali/urea soluble protein,by enzyme linked immunosorbent assay

Summary A sensitive and rapid ELISA for quantitation of rice glutelin was developed. This method employed binding of rice glutelin to Immulon I microtitre plate and quantitating it with commercial goat anti-rabbit IgG conjugate by conventional ELISA. Using this procedure, glutelin as low as 5ng could be detected.     

Novel method for screening pigeonpea varieties for globulin content by radioimmunoassay

Summary A rapid and sensitive radioimmunoassay (RIA) is described for screening a large number of pigeonpea varieties for quantitation of globulin content. This method employs precipitation of iodine-labelled pigeonpea globulin by polyethylene glycol and measuring the labelled antigen-antibody complex. Among the 56 varieties screened, Gwalior-3 was found to contain the highest globulin content. RIA when used to quantitate the pigeonpea globulin levels during post anthesis revealed that maximum globulin is present between 24 to 28 days after flowering.NCL Communication No. 3819.     

DNA sequence organization in the genomes of three related millet plant species

Summary A major portion of the genomes of three millet species, namely, barn yard millet, fox tail millet and little millet has been shown to consist of interspersed repeat and single copy DNA sequences. The interspersed repetitive DNA sequences are both short (0.15–1.0 kilo base pairs, 62–64% and long (>1.5 kilo base pairs, 36–38%) in barn yard millet and little millet while in fox tail millet, only long interspersed repeats (>1.5 kilo base pairs) are present. The length of the interspersed single copy DNA sequences varies in the range of 1.6–2.6 kilo base pairs in all the three species. The repetitive duplexes isolated after renaturation of 1.5 kilo base pairs and 20 kilo base pairs long DNA fragments exhibit a high thermal stability with Tms either equal to or greater than the corresponding native DNAs. The S1 nuclease resistant repetitive DNA duplexes also are thermally stable and reveal the presence of only 1–2% sequence divergence.The present data on the modes of sequence arrangement in millets substantiates the proposed trend in plants, namely, plants with 1C nuclear DNA content of less than 5 picograms have diverse patterns of sequence organization while those with 1C nuclear DNA content greater than 5 picograms have predominantly a short period interspersion pattern.Abbreviations kbp kilobase pairs NCL Communication No. 3606.     

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

DNA fingerprinting detects genetic variability in the pearl millet downy mildew pathogen (Sclerospora graminicola)

Genetic variability in six host genotype-specific pathotypes of pearl millet downy mildew pathogen S. graminicola was studied at the molecular level using mini- and micro-satellites. Our results indicated that microsatellites (GAA)₆, (GACA)₄, and especially (GATA)₄ were quite informative and showed high levels of polymorphism among the pathotypes. The six pathotypes could be classified into five groups based on the cluster analysis of their genetic similarities, thereby confirming the existence of distinct host genotype-specific virulence in S. graminicola pathotypes. We demonstrate, for the first time, the use of DNA fingerprinting to detect genetic variation in downy mildew fungus of pearl millet.     

Genome characterization of glucose-isomerase-producingStreptomyces sp. NCIM 2730: detection of sequences homologous to a rice repeat sequence

Restriction analysis of the genomic DNA from a high glucose/xylose-isomerase-yieldingStreptomyces sp. NCIM 2730 revealed a number of distinct bands on a background smear, indicating the occurrence of repeated DNA sequences in the genome. Optical renaturation analysis indicated that 25% of the genome comprised rapidly reannealing sequences with a copy number of 50 and a kinetic complexity of 3×10³. Hybridization of theStreptomyces genomic library with theStreptomyces DNA, supported the estimate of the repetitive DNA content derived from the re-association kinetics of the DNA. Hybridization of DNA from three differentStreptomyces species with a rice repetitive DNA probe revealed the presence of homologous sequences, which is a unique finding.M.S. Ghatge was and V.V. Deshpande and P.K. Ranjekar are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune -41108, India; M.S. Ghatge is now with the Department of Microbiology & Molecular Biology, The University of Kansas Medical Center, 36th and Rainbow Blvd, Kansas City, Kansas - 66103, USA.