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At least 703 records · Page 39Linked to original sources
PubMed1997

Ehrlichia sennetsu groE operon and antigenic properties of the GroEL homolog.

A clone expressing an immunoreactive 55-kilodalton (kDa) protein of Ehrlichia sennetsu, the causative agent of human Sennetsu ehrlichiosis, was isolated from a gene library of this organism. Sequence analysis of the DNA insert revealed two open reading frames, encoding proteins of 10,620 and 58,225 kDa, respectively. These deduced amino acid sequences were homologous to those of the GroES and GroEL heat shock proteins (HSP) of other bacteria, respectively. Phylogenetic trees based on GroES and GroEL homologs of several bacteria including E. sennetsu showed a relationship similar to that based on 16S rRNA gene sequences. The recombinant and native 55-kDa proteins of E. sennetsu, GroEL homolog, reacted with a monoclonal antibody (SPA807) which recognizes a homologous sequence between human and mycobacterial HSP60 and a polyclonal antibody (SPA804) to cyanobacteria HSP60, but not with antibodies to HSP60 of several other organisms used. Furthermore, anti-recombinant E. sennetsu 55-kDa protein antibody prepared in a rabbit was reactive to HSP60 antigens of other Ehrlichia and Rickettsia species, but not GroEL of E. coli. The recombinant 55-kDa protein would be a useful tool for studying the role of this antigen in the immune response to E. sennetsu infection.

Amino Acid Sequence↗
PubMed2006

Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species.

Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat.

Bacterial Typing Techniques↗
PubMed1986

The cdc22 gene of Schizosaccharomyces pombe encodes a cell cycle-regulated transcript.

Two independent DNA sequences, of 5.6 and 2.4 kb in size, were isolated from Schizosaccharomyces pombe gene libraries on the basis of their ability to rescue the temperature-conditional lethality conferred by a cdc22 mutation. Integration of these sequences into the S.pombe genome by homologous recombination, followed by genetic mapping, demonstrated that the site of integration of the 5.6 kb fragment is tightly linked to the cdc22 locus, while that of the 2.4 kb fragment is unlinked. This shows that the 5.6 kb fragment carries the authentic cdc22 gene while the 2.4 kb fragment carries and extragenic suppressor sequence. The cdc22 transcript was identified by Northern blot analysis and shown to be 3.3 kb in size. The level of the transcript during the cell cycle was investigated in synchronous cultures prepared by elutriation. The cdc22 transcript is cell cycle regulated, reaching a maximum level during late G1/S phase, at least 12-fold higher than the minimum level observed in mid G2.

Journal Article↗
PubMed1995

Schistosoma japonicum: heterogeneity in paramyosin genes.

Paramyosin is an integral muscle protein found in many invertebrates including schistosomes, and is considered an important candidate vaccine antigen in schistosomiasis. The characterisation of natural molecular variation in vaccine antigens including paramyosin is important because strain-specific vaccination may be necessary against schistosomiasis japonica. We have isolated partial cDNAs encoding paramyosin from an adult, Chinese strain Schistosoma japonicum gene library. Two of these cDNAs (B6 and Y6) encode the same region of paramyosin but their nucleotide sequences differ at eight positions and their deduced amino acid sequences differ by an arginine/cysteine substitution, demonstrating intrastrain variation in paramyosin. Southern blot analysis of genomic DNA from the Chinese and Philippine strains of S. japonicum demonstrated strain-related RFLPs at the paramyosin locus, and suggested that more than one copy of the paramyosin gene was present in the S. japonicum genome. PCR-based RFLP analysis which exploited restriction site differences between B6 and Y6 showed that paramyosin genotype B6 was much more common in schistosome populations and verified the existence of introns in the paramyosin gene(s) of S. japonicum.

Amino Acid Sequence↗
PubMed1985

Cloning of a DNA sequence that complements glutamine auxotrophy in Saccharomyces cerevisiae.

Glutamine (gln) requiring mutants of Saccharomyces cerevisiae have been isolated. They synthesize small amounts of glutamine synthetase (GS), which is more thermolabile than the enzyme from the parental strain. The gln auxotrophy was complemented in transformation experiments using an S. cerevisiae gene library constructed in the plasmid vector YEp13. The transformants were mitotically unstable and synthesized almost tenfold higher amounts of GS than wild-type cells. This activity was as thermoresistant as that from the wild-type strain. A recombinant plasmid was isolated from one of the transformants and partially mapped. Upon reintroduction into the auxotrophic strain, the transformation frequency to gln prototrophy was the same as that for the marker LEU2 gene. The evidence presented suggests that we have cloned the structural gene for GS from S. cerevisiae.

Cloning, Molecular↗
PubMed1989

tRNA derived insertion element in histone gene repeating unit of Drosophila melanogaster.

Analysis of 41 histone homologous clones from an isogenic gene library of Drosophila melanogaster showed that non-histone fragments interrupt the histone repetitive clusters at several sites. Long (L) and short (S) forms of the repeating units are distinguished by the insertion of 240 bp into the spacer between H1 and H3 of the L units; Each form appears to be clustered with its own kind. The complete DNA sequence of the histone 5.0 kb repeating unit was determined. Five histone genes (H1, H2A, H2B, H3, H4) were identified in a repeating unit and several sequence blocks common to the five histone genes were found in the 5'- and 3'-regions. The insertion sequence of 240 bp was found to be similar to the Alu family, an element derived from tRNA.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli.

The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell analysis established the subunit M(r) as 43,500. Primer extension analysis indicated the presence of an adequate Pribnow box and suggested that the transcript contained a 110-base leader sequence. Strains harboring the gsk gene on multicopy plasmids overexpressed both guanosine and inosine kinase activities. N-terminal sequence and amino acid composition analyses of the 43,500-M(r) polypeptide band confirmed the correct reading frame assignment and the identity of this band as the gsk gene product. Comparison of the amino acid sequence with the protein database revealed similarity to regions of other mononucleotide-utilizing enzymes.

Amino Acid Sequence↗
PubMed2002

Integrin alpha(v)beta3 targeted therapy for Kaposi's sarcoma with an in vitro evolved antibody.

Here, we define integrin alpha(v)beta3 as a molecular target for antibody therapy for Kaposi's sarcoma (KS). We previously reported, using a new phage display strategy based on designed combinatorial V gene libraries, the humanization of mouse monoclonal antibody LM609 directed to human integrin alpha(v)beta3. In the present study, we describe the in vitro affinity maturation of humanized LM609 by using a phage display strategy for the sequential and parallel optimization of three complementarity determining regions of the antibody molecule. The evolved Fab had an affinity of 150 pM and was converted into IgG1 by use of a new mammalian expression vector. The resulting whole antibody, designated JC-7U IgG1, was found to selectively target human KS in a nude mouse model and inhibit tumor growth at a therapeutically relevant dose. Because of its high affinity and its high degree of humanization, JC-7U IgG1 is an excellent drug candidate for therapeutic applications that involve integrin alpha(v)beta3 as the molecular target. Of particular interest is therapy for KS, breast cancer, melanoma, and other cancers in which integrin alpha(v)beta3 is expressed on both angiogenic endothelial cells and tumor cells, which would allow a dual antiangiogenic and antitumor strike with a single drug.

Animals↗
PubMed1987

[Molecular causes of thalassemia. IV. Cloning of the beta-globin gene in a patient with beta-thalassemia from Azerbaijan and determination of the point mutation in a minor intron].

On the basis of DNA from a beta-thalassemic patient, human gene library has been obtained using bacteriophage lambda EMBL4 as a vector. The recombinants contain human DNA insertions of 15 to 20 kb. The lambda A1 beta 1 clone has been isolated by the method of hybridization of phage plaque replicas to the HhaI fragment of JW102 plasmid containing human beta-globin cDNA. Restriction mapping revealed the presence of a 22 kb human DNA fragment comprising a portion of the beta-globin gene system. Subcloned fragments of beta-globin gene (within the pUC19 plasmid or phage MI3mp10) were sequenced using the Maxam and Gilbert method as well as that of Sanger. 2150 nucleotides in total were analysed. We have detected the point substitution G----A in the 110 nucleotide of minor intron, leading to the formation of an additional splicing site.

Bacteriophage lambda↗
PubMed1983

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.

Actins↗
PubMed2004

Discovery of the novel candidate phylum "Poribacteria" in marine sponges.

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.

Animals↗
PubMed1993

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Amino Acid Sequence↗
PubMed2006

Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis.

The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (>80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found.

Amino Acid Sequence↗
PubMed1986

Cloning of a gene encoding choline transport in Saccharomyces cerevisiae.

By genetic complementation in a yeast choline transport mutant from a yeast gene library, we isolated plasmids encoding choline transport. The cloned plasmids contained a common 4.0-kilobase DNA fragment and also complemented an ethanolamine transport defect. The cloned sequence present in the yeast genome was possibly unique.

Base Sequence↗
PubMed2013

Broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the GP4 or M genes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.

Animals↗
PubMed1988

Functional expression of Aquaspirillum magnetotacticum genes in Escherichia coli K12.

Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro+ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.

Bacteria↗
PubMed2001

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum.

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.

Bacterial Proteins↗