Generation of a Manganese Specific Restriction Endonuclease with Nicking Activity

A typical feature of type II restriction endonucleases (REases) is their obligate sequence specificity and requirement for Mg2+ during catalysis. R.KpnI is an exception. Unlike most other type II REases, the active site of this enzyme can accommodate Mg2+, Mn2+, Ca2+, or Zn2+ and cleave DNA. The enzyme belongs to the HNH superfamily of nucleases and is characterized by the presence of a beta beta alpha-Me finger motif. Residues D148, H149, and Q175 together form the HNH active site and are essential for Mg2+ binding and catalysis. The unique ability of the enzyme to cleave DNA in the presence of different metal ions is exploited to generate mutants that are specific to one particular metal ion. We describe the generation of a Mn2+-dependent sequence specific endonuclease, defective in DNA cleavage with Mg2+ and other divalent metal ions. In the engineered mutant, only Mn2+ is selectively bound at the active site, imparting Mn2+-mediated cleavage. The mutant is impaired in concerted double-stranded DNA cleavage, leading to accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altered reaction conditions. The active site fluidity of R Eases allowing flexible accommodation of catalytic cofactors thus forms a basis for engineering selective metal ion-dependent REase additionally possessing nicking activity.

A proteomic view of probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a probiotic bacterium that is known worldwide. Since its discovery in 1985, the health effects and biology of this health-promoting strain have been researched at an increasing rate. However, knowledge of the molecular biology responsible for these health effects is limited, even though research in this area has continued to grow since the publication of the whole genome sequence of L. rhamnosus GG in 2009. In this thesis, the molecular biology of L. rhamnosus GG was explored by mapping the changes in protein levels in response to diverse stress factors and environmental conditions. The proteomics data were supplemented with transcriptome level mapping of gene expression. The harsh conditions of the gastro-intestinal tract, which involve acidic conditions and detergent-like bile acids, are a notable challenge to the survival of probiotic bacteria. To simulate these conditions, L. rhamnosus GG was exposed to a sudden bile stress, and several stress response mechanisms were revealed, among others various changes in the cell envelope properties. L. rhamnosus GG also responded in various ways to mild acid stress, which probiotic bacteria may face in dairy fermentations and product formulations. The acid stress response of L. rhamnosus GG included changes in central metabolism and specific responses related to the control of intracellular pH. Altogether, L. rhamnosus GG was shown to possess a large repertoire of mechanisms for responding to stress conditions, which is a beneficial character of a probiotic organism. Adaptation to different growth conditions was studied by comparing the proteome level responses of L. rhamnosus GG to divergent growth media and to different phases of growth. Comparing different growth phases revealed that the metabolism of L. rhamnosus GG is modified markedly during shift from the exponential to the stationary phase of growth. These changes were seen both at proteome and transcriptome levels and in various different cellular functions. When the growth of L. rhamnosus GG in a rich laboratory medium and in an industrial whey-based medium was compared, various differences in metabolism and in factors affecting the cell surface properties could be seen. These results led us to recommend that the industrial-type media should be used in laboratory studies of L. rhamnosus GG and other probiotic bacteria to achieve a similar physiological state for the bacteria as that found in industrial products, which would thus yield more relevant information about the bacteria. In addition, an interesting phenomenon of protein phosphorylation was observed in L. rhamnosus GG. Phosphorylation of several proteins of L. rhamnosus GG was detected, and there were hints that the degree of phosphorylation may be dependent on the growth pH.

Interaction of Sesbania Mosaic Virus Movement Protein with VPg and P10: Implication to Specificity of Genome Recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 59 end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.

Analysis of the effects of amino-acid-sequence on the structure of proteins

The conformation of amino acid side chains as observed in well-determined structures of globular proteins has earlier been extensively investigated. In contrast, the structural features of the polypeptide backbone that result from the occurrence of specific amino acids along the polypeptide have not been analysed. In this article, we present the statistically significant features in the backbone geometry that appear to be a consequence of the occurrence of rotamers of different amino acid side chains by analysing 102 well-refined structures that form a random collection of proteins. It is found that the persistence of helical segments around each residue is influenced by the residue type. Several residues exert asymmetrical influence between the carboxyl and amino terminal polypeptide segments. The degree to which secondary structures depart from an average geometry also appears to depend on residue type. These departures are correlated to the corresponding Chou and Fasman parameters of amino acid residues. The frequency distribution of the side chain rotamers is influenced by polypeptide secondary structure. In turn, the rotamer conformation of side chain affects the extension of the secondary structure of the backbone. The strongest correlation is found between the occurrence of g+ conformation and helix propagation on the carboxyl side of many residues.

Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Cloning and sequence of a cDNA encoding a novel hybrid prolme-rich protein associated with cytokinin-induced haustoria formation in Cuscuta reflexa

A complete cDNA encoding a novel hybrid Pro-rich protein (HyPRP) was identified by differentially screening 3x10(4) recombinant plaques of a Cuscuta reflexa cytokinin-induced haustorial cDNA library constructed in lambda gt10. The nucleotide (nt) sequence consists of: (i) a 424-bp 5'-non coding region having five start codons (ATGs) and three upstream open reading frames (uORFs); (ii) an ORF of 987 bp with coding potential for a 329-amino-acid (aa) protein of M(r), 35203 with a hydrophobic N-terminal region including a stretch of nine consecutive Phe followed by a Pro-rich sequence and a Cys-rich hydrophobic C terminus; and (iii) a 178-bp 3'-UTR (untranslated region). Comparison of the predicted aa sequence with the NBRF and SWISSPROT databases and with a recent report of an embryo-specific protein of maize [Jose-Estanyol et al., Plant Cell 4 (1992) 413-423] showed it to be similar to the class of HyPRPs encoded by genes preferentially expressed in young tomato fruits, maize embryos and in vitro-cultured carrot embryos. Northern analysis revealed an approx. 1.8-kb mRNA of this gene expressed in the subapical region of the C. reflexa vine which exhibited maximum sensitivity to cytokinin in haustorial induction.

Interaction of ecoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5?-CAGCAG-3

EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue. We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays. EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly. These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes. In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation. More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide. EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter. Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex. Taken together these results form the basis fora detailed structure-function analysis of EcoP15I DNA Mtase.

Testis-specific histone (H1t) is not phosphorylated and has a weak interaction with chromatin

Soluble chromatin was prepared from rat testes after a brief micrococcal nuclease digestion. After adsorption onto hydroxylapatite at low ionic strength, the histone Hl subtypes were eluted with a shallow salt gradient of 0.3 M NaCl to 0.7 M NaCl. Histone Hlt was eluted at 0.4 MNaCl, while histones H1a and Hlc were eluted at 0.43 M NaCl and 0.45 M respectively. The extreme divergence of the amino acid sequence of the C-terminal half of histone Hlt, the major DNA binding domain of histone Hl, from that of the somatic consensus sequence may contribute to the weaker interaction of histone Hlt with the rat testis chromatin. Further, histone Hlt was not phosphorylated in vivo in contrast to histone Hla and Hlc, as is evident from the observation that histone Hlt lacks the SPKK motif recognized by the CDC-2kinase or the RR/KXS motif recognized by protein kinase A.

The complete primary structure of a unique mannose/glucose-specific lectin from field bean (Dolichos lab lab)

The complete amino acid sequence of two non identical subunits of the glucose/mannose-specific lectin from Dolichos lab lab (field bean) has been determined by sequential Edman analyses of the intact subunits and peptides derived by enzymatic and chemical cleavage. Peptides were purified by reverse phase high performance liquid chromatography and ion pair chromatography. The D. lab lab lectin is a glycoprotein having two polypeptide chains of 132 and 105 amino acid residues. The amino acid sequence of the D. Lab lab lectin is compared with the various lectins of the family Leguminosae. The D. lab lab lectin is the only species of the tribe Phaseoleae that contains two nonidentical subunits of almost equal size and that shows a specificity to glucose/ mannose. The lectin shows a greater homology to the glucose/mannose specific lectins, especially concanavalin A. The unique subunit architecture of the D. lab lab lectin indicates the presence of new post translational cleavage sites.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

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.

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distribution and cytological localisation, Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.

Stochastic dynamics modeling of the protein sequence length distribution in genomes: implications for microbial evolution

In this paper, we report an analysis of the protein sequence length distribution for 13 bacteria, four archaea and one eukaryote whose genomes have been completely sequenced, The frequency distribution of protein sequence length for all the 18 organisms are remarkably similar, independent of genome size and can be described in terms of a lognormal probability distribution function. A simple stochastic model based on multiplicative processes has been proposed to explain the sequence length distribution. The stochastic model supports the random-origin hypothesis of protein sequences in genomes. Distributions of large proteins deviate from the overall lognormal behavior. Their cumulative distribution follows a power-law analogous to Pareto's law used to describe the income distribution of the wealthy. The protein sequence length distribution in genomes of organisms has important implications for microbial evolution and applications. (C) 1999 Elsevier Science B.V. All rights reserved.

Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S-cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfpm2+ gene, coding for GFPm2+ of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.