Characterization of species-specific repeated DNA sequences from B. nigra

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Characterization of human symptomatic rotavirus isolates MP409 and MP480 having ‘long’ RNA electropherotype and subgroup I specificity, highly related to the P6[1],G8 type bovine rotavirus A5, from Mysore, India

In an epidemiological study of symptomatic human rotaviruses in Mysore, India during 1993 and 1994, isolates MP409 and MP480 were isolated from two children suffering from severe, acute dehydrating diarrhea. Both isolates exhibited 'long' RNA pattern and subgroup I specificity suggesting the likelihood of their animal origin. Both isolates did not react with monoclonal antibodies (MAbs) specific for serotypes G1 to G6 as well as CIO. To determine the genetic origin of these isolates, complete nucleotide sequences of genes encoding the outer capsid proteins VP4 and VP7, nonstructural proteins NSP1 and NSP3 and viral enterotoxin protein NSP4 from MP409 and partial sequences of genes from MP480 were determined. Comparison of the 5' and 3' terminal sequences of 250 nucleotides revealed complete identity of the gene sequences in both strains suggesting that MP409 and MP480 are two different isolates of a single strain. Comparison of the nucleotide and deduced amino acid sequences of VP4, VP7, NSP1 and NSP3 of MP409 with published sequences of strains belonging to different serotypes revealed that both outer capsid proteins VP4 and VP7 and NSP1 are highly related to the respective proteins from the P6[1], G8 type bovine rotavirus A5 isolated from a calf with diarrhoea in Thailand and that the NSP3 is highly homologous to that of bovine rotaviruses. The NSP 1 protein showed greatest sequence identity with NSP4s belonging to the KUN genetic group to which NSP4s from human G2 type strains and bovine rotaviruses belong. MP409 and MP480 likely signify interspecies transmission of P6[1], G8 type strains from cattle to humans and represent the first P6[1] type rotaviruses isolated in humans. These and our previous studies on the asymptomatic neonatal strain I321 are of evolutionary and epidemiological significance in the context of close association of majority of the Indian population with cattle.

C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy, Several genes have been identified and cloned from the carrot system, These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using a Cuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes.

Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: Implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14. canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India.

Phylogeny of the freshwater crabs of the Western Ghats (Brachyura, Gecarcinucidae)

The Western Ghats mountain range in India is a biodiversity hotspot for a variety of organisms including a large number of endemic freshwater crab species and genera of the family Gecarcinucidae. The phylogenetic relationships of these taxa, however, have remained poorly understood. Here, we present a phylogeny that includes 90% of peninsular Indian genera based on mitochondrial 16S rRNA and nuclear histone H3 gene sequences. The subfamily Gecarcinucinae was found to be paraphyletic with members of two other subfamilies, Liotelphusinae and Parathelphusinae, nesting within. We identify a well-supported clade consisting of north Indian species and one clade comprising mostly south Indian species that inhabit the southern sky islands' of the Western Ghats. Relationships of early diverging genera, however, were resolved with low support. This study also includes newly sampled material from an isolated mountain plateau in the northern part of the Western Ghats, representing a new species of Gubernatoriana, which we describe here as Gubernatoriana basalticola sp. n. The new species is immediately distinguished from its congeners and the related genera Ghatiana and Inglethelphusa by its carapace and cheliped morphology, which are unique among Indian freshwater crabs. This study highlights the urgent need for continued faunistic studies to assess the true diversity of gecarcinucid crabs on the Indian subcontinent, to fully understand the basal phylogenetic relationships within the freshwater crab family Gecarcinucidae, and to evaluate the conservation threat status and biogeography of the montane freshwater crabs of the Western Ghats.

Effects Of 5' Flanking Sequences And Changes In The 5' Internal Control Region On The Transcription Of Rice Transfer-Rna Gcc Cly Gene

A stretch of 71 nucleotides in a 1.2 kilobase pair Pst I fragment of rice DNA was identified as tRNA~ gene by hybridization and nucleotide sequence analyses. The hybridization of genomic DNA with the tRNA gene showed that there are about 10 glycine tRNA genes per diploid rice genome. The 3' and 5' internal control regions, where RNA polymerase III and transcription factors bind, were found to be present in the coding sequence. The gene was transcribed into a 4S product in an yeast cell-free extract. The substitution of 5' internal control region with analogous sequences from either M13mpl9 or M13mpl8 DNA did not affect the transcription of the gene in vitro. The changes in three highly conserved nucleotides in the consensus 5' internal control region (RGYNNARYGG; R = purine, Y = pyrimidine, N = any nucleotide) did not affect transcription showing that these nucleotides are not essential for promotion of transcription. There were two 16 base pair repeats, 'TGTTTGTTTCAGCTTA' at - 130 and - 375 positions upstream from the start of the gene. Deletion of 5' flanking sequences including the 16 base pair repeat at - 375 showed increased transcription indicating that these sequences negatively modulate the expression of the gene.

Immunological studies on nucleic-acids and their components .6. Antibodies specific to two deoxyribotrinucleotide sequences

Antibodies to the deoxyribotrinucleotides dpApTpA and dpApApT were prepared by injecting the bovine serum albumin conjugates of the respective haptens in rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of the tritiated haptens to the immunoglobulins by various nonradioactive mono- and oligonucleotides, using nitrocellulose membrane binding assay. Anti-dpApTpA and anti-dpApApT antisera were found to contain antibodies which were highly specific to the respective hapten sequence.

Comparative sequence analysis and expression in E-coli of the subgroup I-specific antigen VP6 from a G2 serotype human rotavirus IS2

VP6, the intermediate capsid protein of the virion, specifies subgroup specificity of rotavirus, It is also the most conserved, both at nucleotide and amino acid levels, among group A rotaviruses and is the target of choice for rotavirus detection, In this study we report the sequence of the subgroup I (SGI)-specific VP6 from the serotype G2 strain IS2 isolated from a child suffering from acute diarrhoea in Bangalore ana its comparison with the published VP6 sequences. Interestingly, IS2 gene 6 shared highest homology with that from bovine UK strain and the protein contained substitutions by lysine at amino acid positions 97 and 134, In contrast, the amino acids Met and Glu/Asp at these respective positions are highly conserved in all the other group A rotaviruses sequenced so far, These observations have obvious implications for the evolution of serotype G2 and G2-like strains circulating in India, The SGI VP6, of a human rotavirus, possessing epitopes that are conformationally similar to those found in the native protein in the virion, was successfully expressed in E. coli and purified for the first time by single-step affinity chromatography.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus

A clone showing female-specific expression was identified from an embryonic cDNA library of a mealybug, Planococcus lilacinus, In Southern blots this clone (P7) showed hybridization to genomic DNA of females, but not to that of males, However, P7 showed no hybridization to nuclei of either sex, raising the possibility that it was extrachromosomal in origin, In sectioned adult females P7 hybridized to an abdominal organ called the mycetome. The mycetome is formed by mycetocytes, which are polyploid cells originating from the polar bodies and cleavage nuclei that harbour maternally transmitted, intracellular symbionts. Electron microscopy confirmed the presence of symbionts within the mycetocytes, Sequence analysis showed that P7 is a 16S rRNA gene, confirming its prokaryotic origin, P7 transcripts are localized to one pole in young embryos but are found in the pole as well as in the germ band during later stages of development, P7 expression is detectable in young embryos of both sexes but the absence of P7 in third instar and adult males suggests that this gene, and hence the endosymbionts, are subject to sex-specific elimination. Copyright (C) 1997 Elsevier Science Ltd.