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Biomedical subjects

M Rajagopalan

Publications and source records attributed to M Rajagopalan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination.

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor.

Bacterial Proteins↗
PubMed1996

Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate.

Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped [g]DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-DNA complexes were analyzed by electrophoresis on agarose gels and visualized by autoradiography. The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substrates and from those formed in the absence of gamma-S-ATP. Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP, and the order of preference of binding was gDNA > dsDNA > ssDNA. Since gDNA has both ds- and ssDNA components, we suggest that the role for ATP in RecF protein-DNA interactions in vivo is to confer specificity of binding to dsDNA-ssDNA junctions, which is necessary for catalyzing DNA repair and recombination.

Adenosine Triphosphate↗
PubMed1995

Amplification and cloning of the Mycobacterium tuberculosis dnaA gene.

To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

Amino Acid Sequence↗
PubMed1995

Chimeric receptors expressing juxtamembrane sequences of the insulin receptor undergo rapid endocytosis in the absence of receptor tyrosine kinase activity.

We have examined the endocytosis of chimeric receptors consisting of the extracellular domain of the low density lipoprotein (LDL) receptor linked to the juxtamembrane region of the human insulin receptor (hIR). The latter domain contains amino acid motifs previously shown to be necessary for endocytosis. Here we demonstrate that these codes are also sufficient for normal receptor endocytosis. Chinese hamster ovary cells expressing the chimeric LDL-insulin receptor had internalization and degradation indices of 0.70 +/- 0.01 and 0.51 +/- 0.13 after 5 h at 37 degrees C, compared to 0.62 +/- 0.03 and 0.33 +/- 0.09 for normal LDL receptors. The amino acid sequences that target these chimeras for internalization are the same as those used by the native insulin receptor. The residues GPYL950-953 serve as the predominant endocytosis signal: Mutation of these to APLA led to a 56% reduction in the internalization index. The sequence NPEY957-960 is less important for endocytosis and when mutated to APEA led only to a 32% reduction of chimera internalization. We conclude that the juxtamembrane sequences of the insulin receptor are sufficient to cause internalization of the insulin receptor even in the absence of receptor tyrosine kinase activity.

Animals↗
PubMed1995

A rapid protocol for isolation of RNA from mycobacteria.

Isolation of total cellular RNA from members of mycobacteria has been a labour-intensive task involving large volumes of cells, multiple extractions of cell lysates with phenol-chloroform followed by caesium chloride centrifugation. A simple and rapid procedure is reported for isolation of RNA from mycobacteria using as few as 1 x 10(7) cells. The RNA thus isolated when analysed on ethidium bromide gels contained 16S and 23S RNA as major species. Further, the RNA was used for amplification of an internal segment of hsp65 gene by reverse transcription followed by PCR.

Bacterial Proteins↗
PubMed1995

Mycobacterium smegmatis dnaA region and autonomous replication activity.

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.

Amino Acid Sequence↗
PubMed1995

PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria.

A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone. Single unique patterns were observed with HaeIII and/or BstEII for Mycobacterium fortuitum (100%), M. chelonae (94%), M. abscessus (96%), M. smegmatis (100%), M. mucogenicum (formerly the M. chelonae-like organism) (100%), and the sorbitol-negative third biovariant of M. fortuitum (100%). Evidence is presented in support of two subgroups within M. peregrinum, M. smegmatis, and the unnamed third biovariant of M. fortuitum (sorbitol positive and sorbitol negative). PCR-based technology provides a rapid, accurate system for the identification of clinically important species of RGM which should be particularly useful for reference laboratories.

Bacterial Proteins↗
PubMed1995

Plasma cell balanitis: clinical and histopathological features--response to circumcision.

OBJECTIVE: To evaluate the clinicopathological features and response to circumcision in patients with plasma cell balanitis. SUBJECTS AND METHOD: 32 uncircumcised men with penile lesions typical of plasma cell balanitis. Twenty specimens were available for histopathology. RESULTS: Lesions involved prepuce and glans in 17, prepuce only in 10 and in 5 were localised to glans alone or extended to coronal sulcus. Histopathology showed variable features but were consistent with the diagnosis of plasma cell balanitis. Haemosiderin pigment could be detected in only three specimens of patients with shorter duration of the disease. Twenty seven patients were treated with circumcision and no recurrence was noticed in 3 years of follow up. CONCLUSION: Circumcision is an effective treatment modality in plasma cell balanitis. Absence of haemosiderin pigment in majority of tissue sections is difficult to explain but may be related to longer duration of the disease.

Adult↗
PubMed1994

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

Bacterial Proteins↗
PubMed1992

Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.

Bacterial Proteins↗
PubMed1992

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.

Autoradiography↗
PubMed1991

Amino acid sequences Gly-Pro-Leu-Tyr and Asn-Pro-Glu-Tyr in the submembranous domain of the insulin receptor are required for normal endocytosis.

We have recently shown that the immediately submembranous domain of the human insulin receptor (hIR) is required for rapid ligand-dependent internalization (Thies, R. S., Webster, N. J., and McClain, D. A. (1990) J. Biol. Chem. 265, 10132-10137). This region contains one copy of an NPXY sequence that is required for endocytosis of the low density lipoprotein receptor. In order to dissect and analyze the specific sequences involved in endocytosis of the insulin receptor, we have mutated the NPXY sequence from NPEY (residues 957-960) to APEA (NPEY/APEA). In addition, we have mutated a similar sequence in the same region, changing GPLY (residues 950-953) to APLA (GPLY/APLA). The cDNAs encoding the normal hIR and these mutant receptors were transfected into Rat 1 fibroblasts. The expressed receptors bound insulin with high affinity and retained insulin-stimulated tyrosine kinase activity. Despite the ability of these mutant receptors to bind insulin and undergo autophosphorylation, the GPLY/APLA receptor internalized insulin at only 32% of the rate of normal hIR at low receptor occupancy. On the other hand, the NPEY/APEA receptor internalized insulin at 87% of the normal rate. These results were confirmed by measuring internalization of photoaffinity-labeled insulin receptors. Another receptor with both the NPEY/APEA and GPLY/APLA mutations internalized to a lesser degree than the GPLY/APLA receptor and at a rate equivalent to that seen for a receptor with the entire submembranous domain deleted. A receptor with the complete normal submembranous domain but with the tyrosine kinase and C-terminal region of the hIR deleted exhibited only a basal internalization rate. We conclude that the information contained in the GPLY and, to a lesser extent, the NPEY sequences are necessary but not sufficient for signaling internalization of the insulin receptor.

Amino Acid Sequence↗