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V Subramaniam

Publications and source records attributed to V Subramaniam.

27 records · Page 2Linked to original sources
PubMed1998

Generation of alternative Ultrabithorax isoforms and stepwise removal of a large intron by resplicing at exon-exon junctions.

Little is known about mechanisms that regulate and ensure accurate processing of complex transcription units with long introns. We investigate this in the Ultrabithorax gene of Drosophila. A consensus 5' splice site is regenerated at the junction between the first exon and a small internal exon (mI); this splice site is used in a developmentally regulated manner to remove mI during subsequent processing of the downstream intron. Conserved elements within mI and an interaction with exon mII modulate use of the regenerated splice site. Structural similarities predict the same process for mII. This resplicing mechanism avoids competition between distant splice sites for control of exon inclusion and allows removal of a 74 kb intron as a series of smaller fragments.

Alleles↗
PubMed1998

Cell biological applications of scanning near-field optical microscopy (SNOM).

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical images and is an important technique for visualizing biological systems. We summarize the literature on SNOM of biological systems and present some of our recent applications in cellular biology. These include studies of: i) the binding of fluorescently conjugated lectins to cell surface glycoproteins on 3T3 Balb/c cells, ii) molecular interactions by fluorescence resonance energy transfer using photobleaching techniques, and iii) green fluorescent protein (GFP) expressed in bacteria.

Animals↗
PubMed1997

Evaluation of leptospira micro capsule agglutination test (MCAT) for serodiagnosis of leptospirosis.

A micro capsule agglutination test (MCAT) was evaluated for its usefulness in the diagnosis of leptospirosis. The test was performed on 180 serum samples from 120 patients suspected to have leptospirosis and the results were compared with those obtained by microscopic agglutination test (MAT). The overall sensitivity and specificity of the test in comparison with MAT were 84.7 and 87.0 per cent respectively. Further, to study the relative merits of MCAT and MAT in diagnosing leptospirosis early in the course of the disease, its sensitivity and specificity during the early stages of the disease were compared with those of a single MAT done on acute samples in 60 patients from whom paired samples were available. The test appeared to have a higher sensitivity than MAT during the early stages of the disease (75% vs 58.3%) though the specificity was less than that of MAT (83.3% vs 100%). The sensitivity of the test declined to 61 per cent three to four weeks after the onset of illness. MCAT detected antibodies against serogroups Australis (76.9%), Autumnalis (100%) Ballum (100%), Canicola (100%), Cynopteri (100%), Grippotyphosa (71.8%), Icterohaemorrhagiae (93.3%), Javanica (100%), Pomona (75%) and Pyrogenes (100%). MCAT appears to be a useful screening test for early diagnosis of leptospirosis. It is a simple and easy to read test which does not require any special expertise or equipment.

Agglutination Tests↗
PubMed1996

In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state.

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.

Animals↗
PubMed1996

CD-tagging: a new approach to gene and protein discovery and analysis.

We describe a new method for gene discovery and analysis, CD-tagging, that puts specific molecular tags on a gene, its transcript and its protein product. The method has been successfully tested in two organisms, the haploid unicellular alga Chlamydomonas reinhardtii and the metazoan Drosophila melanogaster. The method utilizes a specially designed DNA molecule, the CD-cassette, that contains splice acceptor and donor sites surrounding a short open reading frame. Insertion of the CD-cassette into an intron in a target gene introduces a new exon, represented by the open reading frame of the CD-cassette, surrounded by two functional hybrid introns. As a result (i) the gene is tagged by a specific nucleotide sequence, (ii) the mRNA is tagged by a specific nucleotide sequence and (iii) the protein is tagged by a specific peptide sequence. Because these tags are unique, specific nucleotide or antibody probes can be used to obtain and/or analyze the gene, transcript or protein. As a gene discovery technology, CD-tagging has two unique advantages: 1) Genes can be identified through a primary screen at the protein level, and so the very process by which a gene is identified provides specific empirical information about its biological function. 2) The cassette arms, which are spliced out of the transcript of the target gene, are available to carry a wide variety of DNA sequences, such as genes encoding drug resistance that can be used to select for the presence of the CD-cassette in the genome.

Animals↗
PubMed1995

Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase.

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, returns to its characteristic native-like value over several days. Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much longer than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.

Alkaline Phosphatase↗
PubMed1994

Functional differences between Ultrabithorax protein isoforms in Drosophila melanogaster: evidence from elimination, substitution and ectopic expression of specific isoforms.

The homeotic selector gene Ultrabithorax (Ubx) specifies regional identities in multiple tissues within the thorax and abdomen of Drosophila melanogaster. Ubx encodes a family of six developmentally specific homeodomain protein isoforms translated from alternatively spliced mRNAs. The mutant allele Ubx195 contains a stop codon in exon mII, one of three differential elements, and consequently produces functional UBX protein only from mRNAs of type IVa and IVb, which are expressed mainly in the central nervous system. Although it retains activity for other processes, Ubx195 behaves like a null allele with respect to development of the peripheral nervous system, indicating that UBX-IVa and IVb alone do not contribute detectable Ubx function for this tissue. The mutant allele UbxMX17 contains an inversion of exon mII. We find that this allele only produces mRNAs of type IVa, but the expression pattern of the resulting UBX-IVa protein is indistinguishable from that of total UBX protein expression in wild-type embryos. The phenotype of homozygous UbxMX17 embryos indicates that UBX-IVa cannot substitute functionally for other isoforms to promote normal development of the peripheral nervous system. This functional limitation is confirmed by a detailed analysis of the peripheral nervous system in embryos that express specific UBX isoforms ectopically under control of a heat shock promoter. Additional observations suggest that UBX isoforms also differ in their ability to function in other tissues.

Alleles↗
PubMedDate not supplied

Fluorescence resonance energy transfer detected by scanning near-field optical microscopy.

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1-10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near-field optical microscope (SNOM) operated in the shared-aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel-by-pixel energy transfer efficiency measurements using SNOM.

3T3 Cells↗