Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.

Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones

The morbilliviruses which infect ruminants, rinderpest (RPV) and peste des petits ruminants (PPRV), are difficult to distinguish serologically. They can be distinguished by differential neutralisation tests and by the migration of the major virus structural protein, the nucleocapsid protein, on polyacrylamide gels. Both these methods are time consuming and require the isolation of live virus for identification; they are not suitable for analysis of material directly from post-mortem specimens. We describe a rapid method for differential diagnosis of infections caused by RPV or PPRV, which uses specific cDNA probes, derived from the mRNAs for the nucleocapsid protein of each virus, which can be used to distinguish unequivocally the two virus types rapidly.

Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones

The apetalal mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower. We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetalal on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation. We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1. We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported. RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1. In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance.

Importance of amino-terminus in maintenance of oligomeric structure of cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1–V30)-SHMT and des-(A1–L49)-SHMT were expressed at very low levels, whereas des-(A1–R58)-SHMT, des-(A1–G75)-SHMT, des-(Q435–F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1–K6)-SHMT and des-(A1–W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 °C, the des-(A1–W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1–K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1–W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1–K6)-SHMT (55 °C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1–W14)-SHMT compared to rSHMT (1.8 ±0.1 M) and des-(A1–K6)-SHMT (1.7 ±0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1–K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1–K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1–W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S–9S poly(A)-containing RNA from rat liver was constructed in Image . Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3′-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.

Nucleotide sequence of the 3' terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins

The 3prime terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3prime terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. (C) 2010 Elsevier B.V. All rights reserved.

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I–III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb) bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. Methodology/Principal Findings: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb)) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB(MtbN)). Gel retardation assays revealed that HupBMtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K-d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HU alpha alpha and HU alpha beta forms. However CTR (C-terminal Region) of HupB(Mtb) imparts greater specificity in DNA binding. HupB(Mtb) protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A: T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. Conclusions/Significance: These data thus point that HupB(Mtb) may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Nucleotide sequence of the 3terminal region of belladonna mottle virus (renamed Physalis mottle virus) RNA and analysis of the evolutionary relationships among tymoviral coat proteins

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addiition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Characterization of species-specific repeated DNA sequences from B. nigra

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.