P.C. Sen Memorial best paper award on rural health practice: A study on visual acuity and vitamin A deficiency among primary school students in Naxalbari Village, Darjeeling district of West Bengal.

331 primary school students studying from Nursery classes to Class IV in 2 randomly selected schools in Naxalbari Village in Darjeeling district of West Bengal were tested for visual acuity (VA), Vitamin A deficiency (VAD) and Bitot's spot. 329 students could be tested for visual acuity whereas all 331 students were tested for VAD and Bitot's spot. The prevalence of abnormal Visual Acuity (VA) (VA < 6/9 in any eye) in this study group was 3.65% and it was highest in 7-8 years age group in both the sexes contributing to 75% of the total students having abnormal VA. All these children fell within 50th percentile of weight and height for their respective age and sex. Hindu & ST children accounted for 91.67% & 50% abnormal VA respectively; however, when presence of abnormal VA was compared with its absence between 2 sexes, Hindu and Muslim students and in different castes, no statistically significant differences were found (p > .05). Students of Class-I and Class-II contributed 25% and 50% respectively and together accounted for 75% of abnormal VA. Prevalence of VAD was 8.16%. Among sufferers of VAD Hindus (81.48%) and ST (51.85%) were the main contributors; the differences between presence and absence of VAD in 2 sexes, among 3 religious groups were not statistically significant (p > .05); however, differences among ST and NonSCST groups, and again SC, ST and Non-SCST groups were statistically significant (p < .05). Prevalence of Bitot's spot was 3.63%. Among the students having Bitot's spot, females (58.33%) outnumber the males (41.67%); but the difference between presence and absence of Bitot's spot in 2 sexes was not statistically significant (p > .05). 83.33% each of Hindus and STs had Bitot's spot. No SC and no Muslim student had this spot; the differences between presence and absence of Bitot's spot between Hindu and Christian students were statistically significant (p < .01); similarly when the findings were considered between ST and NonSCST students the difference was found to be statistically highly significant (p < .001).

Multiple copies of the upstream regulatory region of the major capsid protein gene of bacteriophage MB78 inhibit phage morphogenesis.

The 2.311 kb EcoRI F fragment of bacteriophage MB78 has been cloned in multicopy vectors pUC19 and pCR90. Salmonella typhimurium strains carrying such plasmids cannot support development of phage MB78 while other Salmonella phages like P22 and 9NA grow normally. Most of the phage MB78 induced functions are normal in such transformed hosts but proper maturation of the phage particles does not take place. Deletion of 138 bp from the 3' end of the cloned fragment reverses the inhibitory effect. Analysis of nucleotide and the deduced amino acid sequence of a 1.2 kb HindIII-SalI fragment of the phage genome which overlaps the 138 bp confirms that this part contains the upstream regulatory region of the major structural protein gene. It seems that in presence of multiple copies of the upstream regulatory region (which includes a number of promoter like sequence) of the coat protein gene, the maturase gene is down regulated and this is effective only in cis, a situation quite similar to that of Qbeta RNA phages.

Synthesis of a bacteriophage MB78 late protein by novel ribosomal frameshifting.

MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.

Cloning, sequencing, expression and promoter analysis of a structural protein of bacteriophage MB78.

Bacteriophage MB78, a virulent phage of Salmonella typhimurium isolated in our laboratory. It is different from the well-known temperate phage P22 and 9NA. A detailed physical map has been constructed. To understand more about the physiology and genetics of this interesting phage it has become necessary to fragment the phage genome, clone the fragments and analyze in depth. A number of promoters of bacteriophage MB78 have been cloned and characterized recently. As a part of this program, in this investigation, we report cloning, sequencing and expression and promoter analysis of the ClaI G fragment. We identified the expressed protein as phage structural. Phage structural proteins play a vital role in forming the core head of the phage particle.

Expression of four genes of bacteriophage MB78 from contiguous open reading frames: the genomic organization as deduced by sequence analysis.

Four proteins of bacteriophage MB78 having apparent molecular weights as 35, 14, 21 and 16 kDa are expressed from 3.9 kb SalI-HindIII fragment located almost in the middle of the phage genome. Analysis of the sequence supported by some experimental evidences suggest that these four proteins are expressed from polycistronic message without any intercistronic gap. Stop and start codons of consecutive ORFs overlap and rare initiation codons are used. Computer analysis of the sequence suggests the presence of two more open reading frames within the ORFs of 35 and 16 kDa proteins but in the opposite orientation, i.e. in the complementary strand.

Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78.

Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence. A detailed physical map of this phage has been constructed in our laboratory. In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system. Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system. Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel. The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min. One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5.

Identification of a strong promoter of bacteriophage MB78 that lacks consensus sequence around minus 35 region and interacts with phage specific factor.

A strong promoter of bacteriophage MB78 does not have minus 35 consensus sequence although it has a TGn motif immediately upstream of minus 10 sequence as well as the AT rich UP element. It is efficiently recognised by the sigma 70 RNA polymerase, however, a phage-specific factor competes with sigma 70 RNA polymerase for binding to this region, the binding of the factor being stronger than that of the polymerase. Contrary to the reports in the literature the polymerase appears not to bind to the UP element whereas the phage-specific factor does. The latter seems to be involved in the regulation of the promoter activity.

A supercoil-specific endonuclease from Salmonella typhimurium cleaves both negatively and positively supercoiled DNA.

An enzyme which specifically cleaves supercoiled DNA to linear form through nicked circular form as intermediate was isolated from rifampicin-resistant mutant of Salmonella typhimurium, rif 39. The enzyme activity was stimulated by Mg+2, whereas Ca+2 had no effect. It does not require ATP for its activity. No activity could be detected with relaxed or single stranded circular DNAs. The molecular weight of the enzyme is approximately 34 kDa. The most characteristic feature of this enzyme is that it cleaves both positively and negatively supercoiled DNAs.

Exclusion of temperature bacteriophage P22 by virulent bacteriophage MB78 of Salmonella typhimurium: competition for association with the replication complex.

MB78, a virulent bacteriophage of S. typhimurium does not allow other bacteriophages like P22 and 9NA to multiply in its presence. The exclusion of P22 by MB78 is found to be due to competition for common binding site(s) in the host cell membrane. As a result, P22 DNA fails to replicate in presence of MB78 DNA. Further, the sedimentation profile of P22 DNA in cells infected simultaneously with P22 and MB78 suggested fragmentation of P22 DNA. This may also contribute to the exclusion phenomenon.

Construction of a cloning vector from a naturally occurring plasmid of Salmonella typhimurium.

A naturally occurring plasmid isolated from a drug-resistant strain of Salmonella typhimurium (993) has been used to construct a plasmid vector for cloning in a wild strain of Salmonella. The strain (993) contains at least two plasmids. The smaller plasmid (9 kb) contains an ampicillin-resistant marker, while the larger one (25 kb) is cryptic. Physical mapping of the 9-kb plasmid and construction of a 3.5-kb derivative have been carried out. This plasmid has been used for cloning in a restriction+modification+strain of S. typhimurium using a conventional calcium chloride method. It exhibited better efficiency of transformation than other commonly used plasmids such as pBR322 or its derivatives and transformants were found to be stable in the absence of antibiotic selection. The vector is compatible with pBR322 and can be used to study the expression of cloned genes in minicells.

Abortive infection of the virulent phage 9NA in a fatty acid auxotroph of Salmonella typhimurium: effect of fatty acid supplementation.

A conditional (temperature sensitive) fatty acid biosynthetic mutant (fabB2) of Salmonella typhimurium does not support the development of the virulent bacteriophage 9NA even at permissive temperature (30 degrees C). A limited amount of phage DNA synthesis takes place at this temperature. When the fatty acid composition of the host membrane is altered by growing the cells at 37 degrees C in the presence of exogenous unsaturated fatty acid, differential expression of phage genes was observed. Phage specific lysozyme is induced when the cultures are supplemented with elaidic, palmitelaidic, linoleic and linolelaidic acids but not with oleic and plamitoleic acids. However, in no case were infective particles produced. Under conditions where no lysozyme is synthesized the infected cells increase in length and become filamentous.

Spontaneous deletions of drug-resistance determinants from Salmonella typhimurium in Escherichia coli.

Plasmids isolated from two different clinical isolates of Salmonella typhimurium, both resistant to the antibiotics ampicillin, tetracycline, streptomycin and chloramphenicol, were used to transform Escherichia coli. Segregation of antibiotic-resistance determinants occurred in both cases. Analysis of plasmids from one set of segregants by DNA-DNA hybridisation indicated that the segregation was due to precise deletions in the transforming plasmid.

Isolation of temperature-sensitive mutants of bacteriophage MB78 and correlation between the physical and genetic maps.

In order to study the biochemistry and genetics of the virulent virus MB78 of Salmonella typhimurium, 31 temperature-sensitive mutants of the phage were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. These have been classified into six complementation groups (A through F). Linkage between different complementation groups has been mapped by using two factor crosses between representative members of each group. To correlate the physical and genetic maps of the phage, complementation between bacterial clones carrying plasmids with EcoRI fragments of the phage DNA as inserts and the ts mutants was studied. Good correlation between the physical and genetic maps has been obtained. Tentative locations of the ts mutations on the phage genome have thus been determined.

Hybrid between temperate phage P22 and virulent phage MB78.

Bacteriophage MB78, a virulent phage of Salmonella typhimurium can not grow in rifampicin resistant mutant of the host. However, the temperate phage P22 which grows normally in this host helps MB78 to grow in its non-permissive host. P22 can not itself multiply under the condition of mixed infection and the burst size of MB78 is very much reduced. The burst sizes of both are reduced even when the permissive host LT2 is mixedly infected with P22 and MB78. When rifampicin resistant mutants are mixedly infected, only P22-specific mRNAs are produced in the early stage. This is followed by transcriptions from both P22 and MB78 genomes. Subsequently, only MB78-specific messengers are detected in the infected host. Hybrids between the two phages have been isolated from the mixedly infected cells. These hybrids which contain only 15-20% of P22 genome can grow in the rifampicin resistant mutants of the host. The analysis of hetero-duplex between MB78 and hybrid phages revealed the presence of at least three mismatch regions. Experiments are in progress to identify the parts of P22 genome present in the hybrid phage.

Bacteriophage P22 helps bacteriophage MB78 to overcome the transcription inhibition in rifampicin resistant mutant of Salmonella typhimurium.

Bacteriophage MB78 cannot grow on rifampicin resistant mutant of host Salmonella typhimurium (rif39) which contains an altered beta subunit of RNA polymerase. Bacteriophage P22, however, grows normally in rif39 both in the presence or absence of rifampicin. Perhaps MB78 promoter is not recognized by altered RNA polymerase. As the phage P22 helps MB78 to grow to some extent on rif39, hybrids between P22 and MB78 have been isolated. Hybrid phage which can grow on rif39 contains mostly genes from MB78 although a small portion (15-20 per cent) of the genome belongs to P22 genome which helps MB78 to overcome the transcription inhibition in the host mutant with altered RNA polymerase.

Thiolutin-resistant mutants of Salmonella typhimurium.

Spontaneous mutants of Salmonella typhimurium isolated in our laboratory from thiolutin-containing tryptone agar plates are partially resistant to thiolutin in enriched media. In minimal media, they are not resistant. The mutants are not temperature sensitive but fail to support the development of phage P22 at higher temperatures (40 degrees C). Thiolutin did not interfere with RNA polymerase or nucleotide kinase in in vitro experiments. However, thiolutin did inhibit the rate of incorporation of exogenous uridine into the cellular pool and consequently the acid-precipitable material. It appears that one site of action of thiolutin is at the membrane level.

MB78, a virulent bacteriophage of Salmonella typhimurium.

The isolation and some properties of a virulent bacteriophage of Salmonella typhimurium, MB78, which is morphologically, serologically, and physiologically unrelated to P22, are reported. The phage has a noncontractile long tail with partite ends. It cannot multiply in minimal medium in the presence of citrate. MB78-infected cells are, however, killed in such medium. This phage cannot grow in rifampin-resistant mutants of the host. The latent period of growth of this phage is much shorter than that of P22. Both sieA and sieB genes of the resident P22 prophage are required to exclude the superinfecting MB78 phage, whereas all temperate phages related to P22 are excluded by either one or both of the genes individually. Restriction endonuclease cleavage patterns of P22 and MB78 are distinctly different. The absence of homology between the two phages P22 and MB78 suggests that MB78 is not related to phage P22.

Participation of the host protein(s) in the morphogenesis of bacteriophage P22.

Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40 degree C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.