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S E Hasnain

Publications and source records attributed to S E Hasnain.

At least 55 records · Page 3Linked to original sources
PubMed1995

A multicopy DNA sequence from Meconopsis simplicifolia discriminates between the different species of this endangered Himalayan poppy.

Clones harboring multicopy DNA sequences were isolated on the basis of reverse genome hybridization to Meconopsis paniculata (Himalayan yellow poppy) DNA from a Sau3A partial genomic plasmid library of M. simplicifolia (Himalayan blue poppy). Restriction-endonuclease-dependent genetic polymorphism between five species of Meconopsis, M. aculeata, M. paniculata, M. simplicifolia, M. sinuata and M. villosa, belonging to geographically isolated populations, was evident in genomic DNA filter hybridizations when probed with a clone (pIMS10) isolated from this library. Pooled DNA from seedlings originating from plants of individual populations of M. paniculata, M. simplicifolia and M. villosa gave similar band patterns, with respect to a given enzyme, suggesting intra-population genetic homogeneity.

Cloning, Molecular↗
PubMed1995

The 30-kDa protein binding to the "initiator" of the baculovirus polyhedrin promoter also binds specifically to the coding strand.

We previously reported the purification and characterization of the polyhedrin promoter-binding protein (PPBP), an unusual DNA-binding protein that interacts with transcriptionally important motifs of the baculovirus polyhedrin gene promoter (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S.E. Hasnain, J. Biol. Chem. (1994) 269, 2750-2757. PPBP also exhibits a sequence-specific single-stranded DNA-binding activity. Gel retardations and competition analyses with double- and single-stranded oligonucleotides indicated that PPBP binds the coding strand and not the noncoding strand of the promoter. This was further confirmed by UV cross-linking and Southwestern blotting experiments. Gel retardations with mutated oligonucleotides indicated that both dsDNA and ssDNA binding involve common AATA-AATAAGTATT motifs. However, ssDNA binding is dependent upon ionic interactions unlike dsDNA binding, which is mainly through nonionic interactions. The affinity of PPBP for the coding strand appears to be higher than that for duplex promoter DNA. Interestingly, the PPBP-coding strand complex has a longer half-life (approximately 60 min) than the PPBP-duplex promoter complex (approximately 15 min). PPBP represents a unique example of an "initiator" promoter-binding protein with dual dsDNA and ssDNA binding activities, and this reconciles very well with the unusual binding characteristics displayed by it. The formation of the PPBP-coding strand complex in vivo may be a crucial step for the exceptionally high and repeated rounds of transcriptional activity of the baculovirus polyhedrin gene promoter.

Base Sequence↗
PubMed1995

DNA polymorphism analysis in five endangered species of Meconopsis (Himalayan poppy) using multi-copy sequence-based probes.

An attempted to understand the genetic basis of extinction of Meconopsis (Himalayan poppy), an endangered genus of ornamental value and confined to alpine Himalayas, is described. Multicopy DNA sequences were isolated from a Sau3A partial genomic plasmid library of M. simplicifolia and M. paniculata. Genetic polymorphism between five species of Meconopsis belonging to geographically isolated populations was evident, albeit at a low level, in genomic Southerns when probed with repetitive DNA clones isolated from this library. Intra-population monomorphic band patterns for different populations of a species was evident with respect to a number of enzymes suggesting very low or no genetic variability and/or low mutation rate within a given population of Meconopsis.

Base Sequence↗
PubMed1995

Species-specific 18S rRNA gene amplification for the detection of P. falciparum and P. vivax malaria parasites.

Based on the sequence diversity of the Plasmodium 18S ribosomal RNA (rRNA), we designed oligonucleotide primers for polymerase chain reaction (PCR) to yield different size fragments for P. falciparum and P. vivax. The primers for the PCR procedure were chosen such that the 5' primer was Plasmodium-conserved while the 3' primers were species-specific. Using primer cocktails and cloned plasmid DNAs containing the 18S rRNA genes or parasite genomic DNA as targets, we show that the PCR procedure yields 1.4-kb and 0.5-kb DNA fragments for P. falciparum and P. vivax, respectively. Limited field testing of this procedure demonstrated the utility of a ribosomal gene based species-specific malaria diagnosis.

Animals↗
PubMed1995

Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes.

Codon usage by all the known gene sequences from Autographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (beta hCG) expressed to contrasting levels in the baculovirus system. The highly expressed luc gene showed a codon usage similar to AcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed beta hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base position. Alignment of the 20 nucleotides around the initiation codon of 23 AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system.

Animals↗
PubMed1995

Transcriptional regulation of cell line-dependent, baculovirus-mediated expression of foreign genes.

Cell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinant vAc beta hCG-luc virus carrying two reporter genes--beta subunit of human chorionic gonadotropin (beta hCG) and luciferase (luc)--placed under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived from Spodoptera frugiperda (Sf21 and Sf9), Bombyx mori (BmN and Bm5), and Trichoplusia ni (TN368) were used as host cells. TN368 expressed both beta hCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from the vAc beta hCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Codon usage in the prototype baculovirus--Autographa californica nuclear polyhedrosis virus.

The overall codon usage profile of Autographa californica nuclear polyhedrosis virus (AcNPV) was analyzed, using UWGCG sequence analysis software package from the known protein coding gene sequences available in GenBank Release 72. The analysis revealed that although only 45% of the codon used by AcNPV have G/C at wobble base position, 15 out of 20 AcNPV codons over-utilized for their given amino acids has G/C at the wobble position indicating a possible selection of these codons. The differences in codon usage profile were studied using a parameter called D squared value, calculated with the aid of CORRESPOND program of UWGCG software package. While most of the codon usage profile of the individual genes was very similar to the overall AcNPV codon usage profile (D squared value less than 1.5), there were notable differences (D-squared value greater than 1.5). These genes were polh, p10, ub, sod, gp41, core, 25k, 39k, ie-n, etm, ets most of which, interestingly, belonged to late or very late class and were expressed relatively more efficiently. The two highly expressed genes of AcNPV, polh and the p10, differ from the overall AcNPV codon usage profile with respect to at least nine amino acids (Val, Ala, Ser, Lys, Ile, Thr, Leu, Phe, Arg). Our findings that the two highly expressed late genes polh and p10 utilize a codon usage profile different from the early genes have important implications.

Animals↗
PubMed1994

An unusual 30-kDa protein binding to the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus.

Gel retardation assays using nuclear extracts from the Autographa californica nuclear polyhedrosis virus-infected Sf21 (Spodoptera frugiperda) insect cell line revealed a host factor (polyhedrin promoter-binding protein or PPBP) that binds to the polyhedrin gene promoter. A hexanucleotide sequence (AATAAA) within the promoter is important for binding in association with neighboring elements, some of which are contributed by the sequence TAAGTATT present at the transcription start point. PPBP was affinity purified and appears to be an unusual DNA-binding protein with respect to its stability (binding was obtained at NaCl concentrations and temperatures ranging from 0.2 to 2 M and 0 to 65 degrees C, respectively), high binding affinity (binding even in the absence of nonspecific DNA) with an apparent dissociation constant of approximately 3.7 x 10(-12) M, and high specificity (binding was unaffected in the presence of a 50,000 times excess of nonspecific DNA). From UV cross-linking and Southwestern analyses the molecular mass of PPBP was estimated to be approximately 30 kDa. PPBP is phosphorylated and may have a regulatory function because dephosphorylation abolished DNA binding activity. Differences in PPBP characteristics between nuclear extracts from Sf21 and from Bm5 (Bombyx mori) cell line suggested that additional factors may be involved in the interaction of PPBP with the polyhedrin promoter.

Animals↗
PubMed1994

Beta-subunit of human chorionic gonadotropin hormone and firefly luciferase simultaneously synthesized in insect cells using a recombinant baculovirus are differentially expressed and transported.

A recombinant baculovirus vAc beta hCG-luc was constructed that carried the cDNAs encoding firefly luciferase (luc) and beta-subunit of human chorionic gonadotropin (beta hCG) placed under the transcriptional control of individual copies of the baculovirus polyhedrin gene promoter. The simple, rapid, and sensitive detection of LUC expression was used for selecting recombinant viruses that simultaneously expressed beta hCG, which was identical in all respects to that synthesized using a recombinant baculovirus carrying the beta hCG gene alone. Immunofluorescence staining of virus-infected cells using anti-LUC antibodies revealed that LUC, a nonglycosylated, intracellular protein was retained within the cells whereas beta hCG, an extensively glycosylated, secretory protein, was processed and secreted into the culture medium. LUC and beta hCG were both immunoreactive on Western blot. beta hCG was bioactive, as evident from its ability to associate with alpha hCG and bind with the receptor and produce testosterone in an in vitro mouse Leydig cell assay system. Comparison of recombinant LUC and beta hCG synthesized by the virus-infected insect cells surprisingly revealed that the level of the former was quantitatively higher by at least 10-fold than the latter. A blot of total RNA isolated from vAc beta hCG-luc-infected insect cells, when probed with probes corresponding to the 3' region of the beta hCG or luc genes, indicated differential transcription of the two genes. Computer-aided sequence analysis indicated extensive secondary structure and stem-loop complex-forming potential of the beta hCG gene, which could be responsible for the transcriptional difference observed.

Amino Acid Sequence↗
PubMed1993

Differential secretion and glycosylation of recombinant human chorionic gonadotropin (beta hCG) synthesized using different promoters in the baculovirus expression vector system.

Recombinant baculoviruses vAc beta hCGCOR and vAc beta hCGPOL, carrying the gene (beta hCG) encoding the beta-subunit of human chorionic gonadotropin under the transcriptional control of the late AcNPV core protein gene promoter (PCOR) and the very late polyhedrin gene promoter (PPOL), respectively, were constructed and used to infect lepidopteran cells. Western blot analysis of intra- and extracellular recombinant beta hCG (re-beta hCG) revealed that the secretion of beta hCGCOR was relatively higher. Enzymatic and chemical analysis of carbohydrates showed that beta hCGCOR was more glycosylated than beta hCGPOL. However, the insect-derived beta hCG, with a high-mannose type of sugar, was glycosylated differently and to a lesser extent when compared with the native, urinary beta hCG, and consequently, beta hCGCOR was more bioactive on a unit-mass basis than beta hCGPOL. This temporal gene expression strategy, besides being able to circumvent the 'secretory load' encountered during the synthesis of extensively glycosylated proteins in the baculovirus system, also offers a model to study the role of carbohydrates, both in qualitative and quantitative terms, in protein structure and function.

Animals↗
PubMed1993

Temporal nature of the promoter and not relative strength determines the expression of an extensively processed protein in a baculovirus system.

We demonstrate that the expression of extensively modified and secreted heterologous proteins synthesized in the baculovirus expression vector system (BEVS) depends on the temporal nature of the promoter transcribing the foreign gene. The beta subunit of the human chorionic gonadotropin, an extensively modified secretory glycoprotein hormone was expressed under the transcriptional control of the AcNPV basic protein gene promoter (MP) and the polyhedrin gene promoter (POL), respectively. MP, activated late in the infection cycle, is a weaker promoter when compared to the stronger very late POL promoter. Levels of secretion, immunoreactivity and bioactivity of recombinant proteins, beta hCGMP and beta hCGPOL synthesized under control of the MP and POL promoter were compared. Secretion of beta h CGMP was relatively higher. Enzymatic analysis revealed that the synthesized protein was sialylated. Receptor binding assays and testosterone stimulation assays in a mouse Leydig cell system demonstrated that on a unit protein basis, beta hCGMP was biologically more active than beta hCGPOL.

Animals↗
PubMed1992

Simultaneous synthesis of enzymatically active luciferase and biologically active beta subunit of human chorionic gonadotropin in caterpillars infected with a recombinant baculovirus.

The beta subunit of human chorionic gonadotropin (beta hCG), a secretory and extensively glycosylated hormone, and firefly luciferase, a non-secretory enzyme, were simultaneously synthesized in Spodoptera larvae upon infection with a dual expression recombinant baculovirus, vAc beta hCG-luc. Luciferase was retained predominantly in the body tissue while beta hCG was secreted into the hemolymph of infected larvae. Both the proteins were similar to their authentic counterparts in terms of immunoreactivity and bioactivity. The caterpillar-derived recombinant hCG exhibited reduced electrophoretic mobility on SDS-PAGE and increased biological activity as compared to the hCG expressed in insect cells in culture. The implications of using the larval system for expressing an extensively glycosylated protein are discussed.

Animals↗
PubMed1992

A novel probe for human DNA fingerprinting based on chi-like sequences.

A synthetic oligodeoxyribonucleotide (oligo) containing crossover initiating hotspot-like sequences was designed on the assumption that hypervariability is partly due to the presence of molecular signals which promote recombination. This oligo, when used as a probe for human DNA fingerprinting, generated individual-specific DNA band patterns. The probability of two unrelated individuals having the same DNA band pattern, using this probe, was estimated to be 1.9 x 10(-13).

Base Sequence↗
PubMed1992

Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus.

A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.

Animals↗
PubMed1991

The alpha subunit of human chorionic gonadotropin hormone synthesized in insect cells using a baculovirus vector is biologically active.

A recombinant baculovirus, vAc alpha hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the alpha subunit of hCG was used to express alpha hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 h pi, secreted approximately 11.3 micrograms alpha hCG/2 x 10(6) cells/ml which was identical to the native hormonal peptide in terms of electrophoretic mobility, immunoreactivity and bioactivity on association with beta subunit, as evident by its binding to rat testicular cells and induction of steroidogenesis in a mouse Leydig cell bioassay system. The alpha hCG secreted into the medium represented approximately 20-30% of the total hCG synthesized by vAc alpha CG infected insect cells. The implications of using a very late promoter, in a baculovirus expression system, for directing the transcription of a gene whose gene product requires extensive post-translational modifications are discussed.

Animals↗
PubMed1991

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.

Baculoviridae↗
PubMed1991

Direct in-gel hybridization, without blotting, using nick-translated cloned DNA probe.

Hybridization of DNA and RNA to labeled probes is central to molecular biology. A method is described here for hybridization of cloned DNA probes directly to DNA in agarose gels. This in-gel hybridization method has several advantages over conventional techniques using transfer to membranes. It is extremely rapid, highly sensitive, less expensive and particularly suited for high molecular weight genomic DNA analysis.

Cloning, Molecular↗