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P K Kumar

Publications and source records attributed to P K Kumar.

34 records · Page 2Linked to original sources
PubMed1994

Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis.

We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.

Aniline Compounds↗
PubMed1993

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

Base Sequence↗
PubMed1993

Identification of important bases in a single-stranded region (SSrC) of the hepatitis delta (delta) virus ribozyme.

Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (delta) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.

Base Sequence↗
PubMed1993

Systematic substitution of individual bases in two important single-stranded regions of the HDV ribozyme for evaluation of the role of specific bases.

To elucidate the role of specific bases in the self-cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single-stranded regions [between nucleotides 726-731 (SSrA region) and 762-766 (SSrB region)] were carried out by oligonucleotide-directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base-substitutions and self-cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found to be essential for the ribozyme activity. We could determine the preferred sequences, 5'-G-G-(G/A/U)-N-(A/U/G)-Pu-3' for SSrA and 5'-(G/U)-C-N-(A/G/U)-A-3' for SSrB regions, respectively.

Base Sequence↗
PubMed1993

Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.

Base Sequence↗
PubMed1993

Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme.

In elucidating the part played by the essential stem structures (I, II, and III) in the self-cleavage activity of genomic HDV ribozyme, several point and compensation variants were constructed on pseudoknot-like structure by site-directed mutagenesis. The self-cleavage activities of these variants indicated that stems I and III were essential for the activity by forming Watson-Crick base pairs. On the other hand, disruption of A704:U767 had little influence on the cleavage activity, indicating that it is not essential in forming an active structure. Also, our V1 nuclease probing studies showed that the A704U and HDV88 variants have a sensitivity similar to the nuclease, and major cuts are visible in the stem I and stem II regions. Thus, stem I and stem II regions are maintained together with stem III regions in both molecules. These results and our earlier site-directed mutagenesis studies strongly support a pseudoknot-like structure for the genomic HDV ribozyme.

Base Sequence↗
PubMed1993

Chemical probing studies of the hepatitis delta virus (HDV) genomic ribozyme.

We have investigated the higher-order structure of HDV genomic ribozyme using various base-and phosphate-specific chemical probes under native, semi-denaturing and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethylsulfate and carbodiimide. In general, our results are consistent with the proposed pseudoknot model of secondary structure. The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea.

Base Sequence↗
PubMed1992

Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme.

In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.

Base Sequence↗
PubMed1992

Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions.

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Base Composition↗
PubMed1992

Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation.

Plasmid instabilities in E. coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions. In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions. The selective pressure against pRK248cI stabilized the pMTC48. This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48). In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions. This plasmid was stabilized in the host growing under selective conditions. During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells. Plasmid analysis from the above cells showed that the plasmid has undergone structural instability. Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities. Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.

Base Sequence↗
PubMed1992

Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.

Base Sequence↗
PubMed1992

D-xylose fermentation and catabolism in Fusarium oxysporum.

Fusarium oxysporum exhibits different fermentative capacities when grown under different aeration conditions. Highest ethanol and acetic acid yield coefficients were obtained under semi-aerobic culture conditions. Effect of aerobiosis on the levels of key enzymes and intracellular intermediary metabolites of pentose phosphate pathway, tricarboxylic acid cycle and glycolysis exhibited striking correlation with the fermentative character of metabolism. The results are discussed in relation to the energy levels in the cell, the redox balance and mitochondrial function.

Aerobiosis↗
PubMed1991

Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.

Bacteria↗
PubMed1991

Fusarium oxysporum: status in bioethanol production.

Fermentation of lignocellulosic materials to ethanol and other solvents provides an alternative way of treating wastes and producing chemical feedstocks and fuel additives. Considerable efforts have been made in past 10 years to improve the process based on lignocellulosic biomass and hydrolysate that contains a complex mixture of sugars, decomposition products of sugars, and sometimes the inhibitory levels of soluble lignin. Despite the relative abundance of D-xylose in crop and forest residues it has not been found efficiently fermentable by most of the microorganisms. Recent research has revealed that D-xylose may be fermented to ethanol and organic acids. Recently, several strains of Fusarium oxysporum have been found to have potential for converting not only D-xylose, but also cellulose to ethanol in a one-step process. Distinguishing features of F. oxysporum for ethanol production in comparison to other organisms are identified. These include the advantage of in situ cellulase production and cellulose fermentation, pentose fermentation, and the tolerance of sugars and ethanol. The main disadvantage is the slow conversion rate when compared with yeast.

Biotechnology↗
PubMed1990

Immobilization of genetically engineered cells: a new strategy for higher stability.

The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.

Cloning, Molecular↗