PubMed Health⌕ Search

Biomedical subjects

S E Hasnain

Publications and source records attributed to S E Hasnain.

69 records · Page 4Linked to original sources
PubMed1990

Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo.

Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.

Animals↗
PubMed1990

Expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector.

A cDNA encoding the firefly luciferase [Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was cloned downstream from the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda clone-9 cells. Synthesis of luciferase (Luc) was accurately measured in insect cells growing in a 96-well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. Luc was produced in a coordinate fashion during virus infection. The Luc synthesized in insect cells was not secreted into the medium but was contained within the cell. Our findings suggest that Luc can be used as a superior reporter enzyme for molecular genetic analyses of baculovirus regulatory signals involved in high level expression of foreign genes, protein processing, targeting and stability in insect cells.

Animals↗
PubMed1990

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism.

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.

Base Sequence↗
PubMed1989

Nucleotide sequence of cloned nad4 (urf4) gene from Chlamydomonas reinhardtii mitochondrial DNA.

Chlamydomonas reinhardtii mitochondrial (mt)DNA was digested with ClaI + HpaI and shotgun cloned into the M13mp19 vector cleaved with AccI + SmaI. One of the recombinant clones, with a 1.8-kb DNA insert, was completely sequenced using the dideoxy chain-termination method. Besides containing part of the cytochrome b (COB)-encoding gene (cob), this DNA fragment encodes subunit 4 of NADH dehydrogenase (NAD4). The deduced amino acid sequence and hydrophilicity plot indicate that NAD4 is highly hydrophobic. The nad4 gene shows a unique preference for certain codons which are also found in other C. reinhardtii mt proteins. Both the genes encoding NAD4 and COB are shown to be transcriptionally active by Northern hybridization. These closely linked genes suggest that RNA-processing events found in vertebrate mt are present in Chlamydomonas mt as well.

Amino Acid Sequence↗
PubMed1988

DNA-mediated transformation in the aquatic filamentous fungus Achlya ambisexualis.

A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2 microns. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the transformant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.

Chytridiomycota↗
PubMed1987

Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis.

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.

Chytridiomycota↗
PubMed1985

DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker.

Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3'-phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick-blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant.

Chlamydomonas↗
PubMedDate not supplied

Molecular genetic analysis of multi-drug resistance in Indian isolates of Mycobacterium tuberculosis.

A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo beta, gyr A, kat G were studied for mutation(s) by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique. The SSCP positive samples were sequenced to characterize the mutations in rpo beta, and gyr A loci. While previously reported mutations in the gyr A and rpo beta loci were found to be present, several novel mutations were also scored in the rpo beta locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s) that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR) in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis from India, and highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

Humans↗