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At least 415 records · Page 23Linked to original sources

PubMed1995

Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai.

Aplysianin-A, an antibacterial glycoprotein in the albumen gland of the sea hare Aplysia kurodai, inhibited the growth of both Gram-positive and Gram-negative bacteria. Aplysianin-A cDNA clones were isolated from an albumen gland cDNA library. Sequence analysis reveals that aplysianin-A is produced as a precursor protein of 556 amino acid residues with a signal peptide of 19 amino acid residues and contains 6 potential N-glycosylation sites. Aplysianin-A mRNA was expressed tissue-specifically in the albumen gland. Homology search reveals that aplysianin-A has a 50% overall amino acid sequence homology to achacin, an antibacterial glycoprotein of the giant African snail Achatina fulica.

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster.

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.

Animals↗

PubMed1989

Recruitment to the cytoplasm of a cellular lamin-like protein from the nucleus during a poxvirus infection.

Monoclonal antibodies (Mabs) directed against core proteins of rabbit poxvirus (RPV) have proven effective in the identification of host cell proteins such as RNA polymerase II (Pol II) that may play a role in the infectious process (D. K. Morrison and R. W. Moyer, 1986, Cell 44, 587-596). In this article we describe a Mab that has allowed the detection and characterization of a lamin-like protein derived from the nucleus of the infected cell, which like Pol II is recruited to the cytoplasm following RPV infection. A portion of the gene encoding this protein has been isolated through the screening of a lambda gt11 expression vector library. Sequence analysis of the gene shows it to be derived from a member of the HindIII 1.9-kb repetitive element, a family of mammalian repetitive sequences that are highly conserved. Immunoblot analysis and sequence analysis of the open reading frame show divergent relatedness to certain nuclear lamins. The protein is not, however, one of the three principal lamins characterized to date, but instead appears to be a perinuclear protein related to the highly conserved nuclear lamins that is recruited to the cytoplasm during the infectious process.

Amino Acid Sequence↗

PubMed1991

Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of cDNA clones encoding the skeletal and smooth muscle Xenopus laevis beta tropomyosin isoforms.

cDNAs clones corresponding to the skeletal and smooth muscle beta tropomyosins isoforms were isolated from a Xenopus laevis embryo cDNA library. Sequence analysis indicated that the two isoforms are coded by a single gene that uses two couples of alternative exons. The expression of the X. laevis beta tropomyosin gene closely resembles that of the mammalian gene but differs from the avian gene.

Amino Acid Sequence↗

PubMed1995

Several splicing variants of isl-1 like genes in the chinook salmon (Oncorhynchus tschawytscha) encode truncated transcription factors containing a complete LIM domain.

Several novel cDNA clones have been isolated from a chinook salmon pituitary cDNA library. Sequence analysis of these clones indicates that they are closely related to the rat LIM domain homeobox gene, isl-1. Due to differential splicing, several of the clones encode truncated polypeptides containing a complete copy of the LIM domain without the homeodomain and C-terminal activation domain. The roles of these truncated polypeptides are discussed.

Amino Acid Sequence↗

PubMed1995

Cloning and characterisation of a fish aldolase B gene.

A full length cDNA clone representing an aldolase mRNA was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone revealed it to encode a 364 amino acid protein with 74% amino acid identity to human aldolase B and slightly lower similarity to human aldolase A and C. In view of the sequence data and of Northern blot analysis showing strong expression of a 1.6 kb transcript in liver it was concluded that the cloned gene represents aldolase B. This clone represents the first aldolase gene to be sequenced from any fish species thus providing new data on the evolution of the vertebrate aldolase gene family.

Amino Acid Sequence↗

PubMed1995

Deer antler tissue contains two types of bone morphogenetic protein 4 mRNA transcripts.

Previously we isolated a bone morphogenetic protein 4 (BMP-4) cDNA from human prostate cancer cells and found that the 5' noncoding exon 1 of this BMP-4 cDNA was different from that of human bone cell BMP-4 cDNA. Recently we identified two alternate exon 1s, 1A and 1B, for BMP-4 gene by reverse transcription-polymerase chain reaction (RT-PCR) assays from fetal rat calvarial osteoblasts. In order to further examine alternate exon 1 usage in the BMP-4 gene, we screened deer antler tissue cDNA library. We isolated two types of cDNA clones encoding BMP-4 from this deer antler cDNA library. Sequencing of these clones have revealed a single open reading frame encoding a 408 amino acid protein. Comparison of 5' noncoding exon 1 portion of these cDNA sequences with those of human bone and prostate BMP-4 cDNA sequences and mouse BMP-4 genomic DNA sequence demonstrated that deer antler tissue expresses both exon 1A and 1B containing BMP-4 mRNA transcripts. This suggests that BMP-4 gene may contain alternate promoters or alternate splicing sites in deer antler tissue.

Amino Acid Sequence↗

PubMed1994

Cloning and expression of a cDNA encoding bovine muscarinic acetylcholine m3 receptor.

A cDNA clone encoding bovine muscarinic acetylcholine m3 receptor has been isolated from a bovine brain library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 590 amino acids. Comparison with known muscarinic m3 receptor sequences from other species shows a high degree of conservation (92-98% homology) in the protein sequence. COS cells transfected with this cDNA expressed a single high-affinity (Kd = 43.9 pM) binding site for muscarinic receptor ligand N-[3H]methylscopolamine.

Amino Acid Sequence↗

PubMedDate not supplied

The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.

Bacillus subtilis↗

PubMed1995

cDNA cloning and chromosomal localization of the human ciliary neurotrophic factor gene.

Full-length cDNA for human ciliary neurotrophic factor (CNTF) was isolated from a human sciatic nerve cDNA library. Sequence analysis revealed that the longest cDNA was comprised of a 48-bp 5'-untranslated region, a 600-bp coding region and a 1207-bp 3'-untranslated region containing four ATTTA pentamer motifs and a polyadenylation sequence. The transcription starting point was assigned at 81 bp upstream of the initiation methionine by 5' RACE analysis. Using the cDNA and genomic DNA fragment including the entire intron region as mixed probes, the human CNTF gene was localized to the long arm of chromosome 11 at region q12 by fluorescence in situ hybridization.

Chromosomes↗

PubMed1989

Cloning and analysis of a human 86-kDa heat-shock-protein-encoding gene.

An 86-kDa heat-shock-protein-encoding (hsp86) cDNA probe permitted to identify, in whole genomic human DNA, two EcoRI fragments of 2.6 and 5.3 kb. These two fragments, as well as an homologous phage lambda VIII1 harboring about 19 kb of human DNA, were isolated from genomic libraries. Sequence analysis revealed that three different genomic hsp86 sequences had been cloned, one of them being the 5' half of a functional gene. This gene contains several introns, as compared to the entire Hsp86-encoding sequence found in lambda VIII1, which represents a processed pseudogene. Cloned hsp86 promoter, with its TATA-box and a heat-shock element upstream at nt positions -25 and -75, respectively, was functional, as verified by fusion to the bacterial chloramphenicol acetyltransferase-encoding gene and its transient expression in vivo. The typical hsp86-type heat-shock regulation was observed, i.e., significant basal activity associated with an inducibility at elevated temperatures. Furthermore, accurate and efficient in vitro transcription was initiated at this hsp86 promoter, resulting in expression of the hsp86 gene, as well as the unrelated sequences.

Amino Acid Sequence↗

PubMed1995

Structure and gene expression of avian cyclin D2.

Avian cyclin D2 (Cyl D2)-encoding cDNA clones were isolated from a chicken UG9 T-cell lambda gt10 library. Sequence analysis revealed a high degree of sequence conservation with both the mouse and human Cyl D2, and somewhat lower similarity with the mouse and human Cyl D1 and D3. The homology is highest between species in the Cyl-box domain which is well conserved among human, mouse and chicken. A single 6.0-kb CYL2 mRNA is produced in both avian B- and T-cells, as expected.

Amino Acid Sequence↗

PubMed1996

Processing and expression of rat and human clotting factor-X-encoding cDNAs.

The cDNA encoding clotting factor X, which participates in the middle stage of the blood coagulation cascade was cloned from a rat liver cDNA library. Sequencing of the rat factor-X-encoding cDNA revealed that this vitamin-K-dependent protein has a dibasic Arg-Arg sequence at the propeptide cleavage site, as occurs in other vitamin-K-dependent proteins. Although the human and rat deduced amino acid sequences are remarkably similar (76% identical), they do significantly differ in that human factor-X contains a unique Thr-Arg sequence at the propeptide cleavage site [Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985) 3591-3595], where a dibasic sequence would normally be expected. This specific site is the recognition motif for the endoprotease, furin, which is located in the Golgi apparatus. Both rat and human cDNAs expressed in Cos-1 cells resulted in secretion of a mixture of single- and two-chain forms of factor X. The two-chain forms were devoid of the propeptide and were produced at similar rates by the transfected cells. The efficient processing of human factor X, when compared to rat factor X, may indicate that an additional protease(s), which recognizes the Thr-Arg motif, may be involved in proteolytic processing of the human enzyme.

Amino Acid Sequence↗

PubMed1989

Isolation and chromosomal localization of the human En-2 gene.

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.

Amino Acid Sequence↗

PubMed2005

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum.

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold (P < 0.01) between days 13 and 22 of pregnancy and 8-fold (P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Amino Acid Sequence↗

PubMed2026

CRISPGen: A deep generative framework for multi-objective CRISPR/Cas9 guide RNA design via Conditional Latent Diffusion and Dual-Critic Reinforcement Learning.

MOTIVATION: The CRISPR-Cas9 system offers transformative potential for precision genome editing, yet its clinical translation remains constrained by the risk of unintended off-target double-strand breaks. While current discriminative models excel at evaluating pre-specified candidate guides, resolving the fundamental antagonism between on-target cleavage efficiency and off-target specificity within a fixed sequence search space remains a major challenge. RESULTS: We present CRISPGen, a unified deep generative framework that reframes sgRNA design as a multi-objective constrained sequence synthesis problem. It integrates (i) DNABERT-2 genomic-language embeddings, (ii) a conditional latent diffusion generator conditioned on a user-specified on-target efficiency target, and (iii) a dual-critic reinforcement-learning (RL) stage that couples a frozen on-target efficiency critic with a cross-attention off-target discriminator (validation Pearson R=0.8157) trained on a unified corpus of experimental off-target events from six detection platforms. Across 1000 generated sgRNAs, CRISPGen reduces the mean off-target discriminator score by 99.7% relative to the pre-RL baseline and, under an exhaustive whole-genome screen of all 302,631,056 NGG PAM sites in GRCh38, yields zero perfect-match and only 55 one-mismatch genomic hits. We further show, transparently, that the internal on-target critic saturates under RL optimization - an instance of Goodhart's Law - and therefore assess on-target viability using an independent external CRISPRon screen (mean 47.10/100). Repeating the RL fine-tuning stage under three random seeds (with the diffusion generator, DNABERT-2 embeddings, and off-target discriminator held fixed) yields a stable operating point across seeds. Full diversity, per-mismatch, and reproducibility statistics are reported in the Results. AVAILABILITY: Source code is available at https://github.com/malekpouri/CRISPGen; the pre-trained checkpoints and the 3,000,000-sequence library are hosted on Hugging Face (https://huggingface.co/malekpouri/CRISPGen-Checkpoints) and archived on Zenodo under DOI 10.5281/zenodo.21428641.

CRISPR-Cas9↗

PubMed2006

Rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization.

We report the identification of immune-related molecules from the fat body, and intestine of Rhodnius prolixus, an important vector of Chagas disease. Insects were challenged by introducing pathogens or Trypanosoma cruzi, the parasite that causes Chagas disease, into the hemocoel. RNA from intestines, or fat body were isolated 24h after stimulation. We used suppressive subtractive hybridization to identify immune-related genes, generated three subtracted libraries, sequenced the clones and assembled the sequences. The functional annotation revealed expressed sequence tags (ESTs) generated in response to various stimuli in all tissues, and included pathogen recognition molecules, regulatory molecules, and effector molecules.

Animals↗