Cloning and characterization of a β-galactosidase encoding region in Lactobacillus coryniformis CECT 5711

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional β-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric β-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another β-galactosidase, an α-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis β-galactosidase was overproduced in E. coli by using an isopropyl-β-d-thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M _r s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed β-galactosidase activity.     

Cloning and characterization of zebrafish protocadherin–17

Protocadherins constitute the largest subgroup within the cadherin superfamily of cell surface molecules. In this study, we report the molecular cloning and expression analysis of the non-clustered protocadherin-17 (pcdh17) in the embryonic zebrafish nervous system. The zebrafish Pcdh17 protein is highly conserved, exhibiting 73% sequence homology with the human protein. The zebrafish pcdh17 gene consists of four exons spread over 150 kb, and this organization is highly conserved throughout vertebrates. Pcdh17 message is first detectable by 6 h postfertilization in the developing embryo, and the expression is maintained throughout development. Zebrafish embryos express pcdh17 in all of the major subdivisions of the central nervous system, including the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Analysis of the genomic sequence upstream of pcdh17 in several species reveals a pattern of paired CpG islands. While the CpG islands in zebrafish are further upstream than in other teleosts, alignment of the identified sequences reveals a high degree of conservation, suggesting that the sequences may be important for the regulation of pcdh17 expression.     

Isolation and characterization of phage–host systems from the Baltic Sea ice

In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.     

Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.     

Cloning and characterization of a Δ9-desaturase gene of the Antarctic fish Chionodraco hamatus and Trematomus bernacchii

Chionodraco hamatus and Trematomus bernacchii are perciforms, members of the fish suborder Notothenioidei that live in the Antarctic Ocean and experience very cold and persistent environmental temperature. These fish have biochemical and molecular features that allow them to live at these extreme cold temperatures. Fine tuning of the level of unsaturated fatty acids content in membrane is a key mechanism of living organisms to adapt to cold and high temperatures. Desaturases are key enzymes that synthesize unsaturated fatty acyl-CoAs from saturated fatty acids. We cloned and sequenced a Δ⁹-desaturase gene and its cDNA of C. hamatus, and the cDNA of T. bernacchii. The coded proteins are virtually identical and share homology to other Δ⁹-desaturase fish sequences. These proteins contain, in the first trans-membrane domain, two cysteine residues that may form a disulfur bond present in the corresponding membrane region of Δ⁹-desaturase proteins of other Antarctic fish but not in Eleginops maclovinus that experiences higher environmental temperatures and in all other Δ⁹-desaturase genes of mammals present in data bases. C. hamatus Δ⁹-desaturase gene complements a Saccharomyces cerevisiae mutant lacking Δ⁹-desaturase (Ole1) gene. Analysis of sequence homology of the trans-membrane domains of Δ⁹-desaturase and the cytoplasmic region of the same proteins of Antarctic fish, non-Antarctic fish and mammals suggest that the significant differences found in the homologous sequences of the first trans-membrane domain may be due to the specific lipid content of their membrane.     

Cloning and characterization of the BglII restriction–modification system reveals a possible evolutionary footprint

BglII, a type II restriction–modification (R–M) system from Bacillus globigii, recognizes the sequence 5′-AGATCT-3′. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R–M system. Oligonucleotide probes specific for the 5′ end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R–M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R–M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R–M system.     

The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.     

Cloning and analysing of 5‘ flanking region of Xenopus organizer gene noggin

Xenopus organizer specific gene noggin possesses nearly all the characterestic properties of the action of organizer to specify the embryonic body acis.To analyze how the maternal inherited factors control its expression pattern,we cloned the 5‘ regulatory region of noggin gene.The 1.5 kb upstream sequense could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragmet is essential for activating noggin expression.We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components.     

Albinism and immunity: what's the link?

A small number of inherited diseases show a combination of immunological and pigmentation defects. Chediak-Higashi, Griscellis and Hermansky-Pudlak syndromes are all autosomal recessive diseases with these characteristics. Recent advances in both the identification of the genes giving rise to these diseases and the cell biology of immune cells and melanocytes have begun to reveal the molecular links between immunodeficiencies and albinism. These studies identify key proteins, such as Rab27a, which are critical for secretion of specialised granules found in melanocytes and immune cells. The granules of these cells are modified lysosomes termed 'secretory lysosomes'. These studies reveal that secretory lysosomes use specialised mechanisms of secretion, not found in other cell types, which explains the selective defects in these diseases.     

Cloning of the human α2-macroglobulin gene and detection of mutations in two functional domains: the bait region and the thiolester site

Summary Overlapping genomic clones of the human ₂-macroglobulin (2M) gene were isolated from a cosmid library and were used to map 80 kb of the chromosomal region of this gene. Fragments carrying the two exons encoding the bait region and the exon encoding the thiolester site were partially sequenced and PCR primers were designed for the amplification of both functional domains. By direct genomic sequencing of these domains in 30 healthy individuals and in 30 patients with chronic lung disease three mutations were detected. The first was a sequence polymorphism occurring near the thiolester site of the gene, changing Val¹⁰⁰⁰(GTC) to Ile¹⁰⁰⁰(ATC), with allele frequencies of 0.30 (GTC) and 0.70 (ATC), respectively. No difference of 2M serum levels was observed for these two alleles. The second mutation occured within the thiolester site of one patient, changing Cys⁹⁷²(TGT) to Tyr⁹⁷²(TAT). Since activation of the internal thiolester formed between Cys⁹⁷² and Gln⁹⁷⁵ in each of the subunits of the tetrameric 2M is involved in the covalent cross-linking of the activating proteinase, this mutation is predicted to interfere with 2M function. The 2M serum level was within the normal range in this patient. In one healthy individual we detected an alteration of the bait region sequence, which is usually encoded by two different exons separated by an intron of size 1.6kb. In this individual, PCR amplification of genomic DNA using the bait region primers produced the common fragment of size 1.8 kb and an additional variant fragment of size 0.23kb. This finding, and the genomic sequencing data of this individual, indicate that he carries two different alleles of the 2M gene: one with the regular structure (bait exon I-intron-bait exon II), the other with the two bait exons fused into one. Direct genomic sequencing of the two 2M functional domains is a useful tool for the detection of the genetic, and possibly the functional, heterogeneity of 2M. This, in turn, may provide some insight into the hitherto unknown physiological role(s) of 2M, by studying in vivo effects of naturally ocurring mutations of the gene.     

Cloning and expression of a β-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme

A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a -galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a -glycosidase (tt-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Tt-gly showed strong identity with those of -glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of -D-galactoside, -D-glucoside and -D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl--D-glucoside and p-nitrophenyl--D-fucoside than for p-nitrophenyl--D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: 1-3 (100%) < 1-2 (71%) < 1-4 (40%) < 1-6 (10%). Tt-gly is a thermostable enzyme displaying an optimum temperature of 88°C and a half life of 10 min at 90°C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.     

Isolation and characterization of deletion mutants affected in early genes of bacteriophage ?80

Summary When Escherichia coli cells that had been irradiated with ultraviolet light were infected with bacteriophage 80, five major (pE, pB, pA, pC and pD) and two minor (pU and pV) proteins were found to be synthesized during early stages of infection. The genss coding for the five major proteins were mapped on the 80 chromosome using various deletion mutants which lacked the capacity to synthesize some or all the major proteins. The size and positions of all the deletions were determined by gel electrophoresis of EcoRI digests of phage DNA and by electron microscopy of heteroduplexes between DNAs of the deletion and wild-type phage. The five major proteins designated pE(25K), pB(40K), pA(45K), pC(34K) and pD(31K) were shown to be encoded in this order presumably by a single operon that was located at 60.2–67.4% on the 80 genome. These proteins were found to be involved in phage recombination. The absence of pE or pB resulted in a Red^– phenotype and the absence of three proteins (pE, pB and pA) resulted in a Fec^– phenotype. The exact positions of the genes for the minor proteins pU(29K) and pV(26K) have not been determined.     

Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin

Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37 °C, pH 7.5 and concentration of NaCl not higher than 150 mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4–0.8 OD_600 nm s⁻¹ mg⁻¹). In storage conditions (4 °C and 22 °C, pH 6.0–9.0, 10–500 mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4 °C, pH 6.0–7.5, 10 mM NaCl) were selected under which the time of the enzyme half-inactivation is 120–160 days. LysPhi80α stability is insufficient: at 37 °C the enzyme loses half of its activity almost immediately; at 4 °C and 22 °C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.