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At least 433 records · Page 24Linked to original sources

PubMed1991

Heavy metals induce expression of the TPA-inducible sequence (TIS) genes.

We previously cloned a set of primary response genes, which we call TIS (TPA-Inducible Sequence) genes, from a cDNA library prepared from Swiss 3T3 cells treated with tetradecanoyl phorbol acetate (TPA) and cycloheximide. TPA, polypeptide growth factors, and serum induce TIS gene expression in 3T3 cells. We now report that cadmium and zinc elevate mRNA levels for the TIS genes, including TIS28 (c-fos), in Swiss 3T3 cells. The time-course of TIS gene mRNA accumulation after metal exposure is delayed in comparison to the accumulation of TIS gene mRNA after treatment with TPA and growth factors. Cadmium induction of the TIS gene message accumulation is blocked by actinomycin D. Moreover, cadmium treatment does not significantly stabilize TIS gene messages. TIS gene induction by metal is a primary response; TIS8, which encodes a zinc-finger transcription factor, and TIS28 (c-fos) can be induced in the presence of cadmium and cycloheximide, an inhibitor of protein synthesis. Down-regulation of protein kinase C does not attenuate TIS gene induction by heavy metals.

Animals↗

PubMed2025

ATAC-seq in Emerging Model Organisms: Challenges and Strategies.

The Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a versatile and widely utilized method for identifying potential regulatory regions, such as promoters and enhancers, within a genome. ATAC-seq has been successfully applied to a wide range of established and emerging model organisms. However, implementing this method in emerging model systems, such as arthropods, can be challenging due to several factors that influence data quality. These factors include the availability of a sufficient amount and quality of tissue or cells, the need for species- and tissue-specific protocol optimization, the completeness and accuracy of the reference genome, and the quality of the genome annotation. In this article, we emphasize the key steps in the ATAC-seq protocol that, based on our experience, have the greatest impact on data quality when adapting this method for emerging model organisms. Specifically, we discuss the importance of nuclei isolation, the incubation conditions of the Tn5 transposase, and PCR amplification of the library. Furthermore, we outline essential quality checkpoints during the bioinformatic analysis of ATAC-seq data to assist in assessing data integrity and consistency. Given that many emerging model organisms may not be readily available in laboratory cultures, we also emphasize the importance of evaluating how different preservation methods affect ATAC-seq data quality. Based on examples in one spider and one ant species, we demonstrate that replication and thorough quality controls at all steps of the protocol and data analysis are essential to assess the usability of ATAC-seq data. Our data highlights the importance of isolating the right number of intact nuclei, as well as ensuring optimal amplification conditions during library preparation to obtain good-quality sequence data for downstream analyses. We recommend using fresh tissue samples if possible because we show that direct cryopreservation of the tissue may affect chromatin integrity. This effect could be avoided or reduced by preserving the homogenate in cell culture medium. Overall, we explain the ATAC-seq protocol and downstream analyses in detail and give step-by-step advice to researchers who are new to the field and want to implement this method. With careful planning and validation, ATAC-seq can reveal the regulatory landscape of a genome and aid in identifying elements that govern gene expression.

Animals↗

PubMed2003

Positive Darwinian selection operating on the immunoglobulin heavy chain of Antarctic fishes.

The cooling of the Southern Ocean to the freezing point of seawater (-1.9 degrees C) over the past 25 million years played a dominant selective role in the evolution of the Antarctic fish fauna. During this period, the perciform suborder Notothenioidei, which is largely endemic to the Antarctic, diversified and developed numerous cold-adapted characters. In this report, we provide compelling evidence that the immunoglobulin heavy chain (IgH) of the notothenioid fishes has undergone adaptive selection. Two and four IgH clones were isolated, respectively, from spleen cDNA libraries prepared from the Antarctic icefish Chaenocephalus aceratus and the yellowbelly rockcod Notothenia coriiceps. The transmembrane region of the membrane form of the rockcod IgM heavy chain was located at the end of the second constant (C(H)) domain, in contrast to other teleost IgMs in which the transmembrane region is located at the end of the third constant domain. Phylogenetic analyses of C(H) regions revealed that rates of nonsynonymous nucleotide substitution were higher than rates of synonymous nucleotide substitution. Many of the nonsynonymous substitutions introduced charge changes, consistent with positive Darwinian selection acting to adapt the structure of the notothenioid immunoglobulins. The rates of nonsynonymous nucleotide substitutions were higher than the rates of synonymous nucleotide substitutions in complementarity determining regions of variable regions, suggesting that diversity at antigen binding sites is enhanced by genomic and/or somatic selection. Results of Southern blot hybridization experiments were consistent with a translocon type of IgH gene organization reminiscent of bony fishes and tetrapods.

Adaptation, Physiological↗

PubMed1989

Identification and cDNA cloning of a new member of the L2/HNK-1 family of neural surface glycoproteins.

Rabbit antibodies raised against a 135- to 140-kD glycoprotein isolated from the culture medium of mouse forebrain explants were used for the identification and cloning of a complex of mouse neural cell surface glycoproteins. The antibodies recognized a 135-kD surface protein which shared the L2/HNK-1 epitope with several neural cell adhesion molecules. Three homologous complementary deoxyribonucleic acid (cDNA) clones were isolated from a mouse brain cDNA library prepared in the expression vector lambda gt11, one of which was sequenced and found to lack sequence homologies with known proteins. In Northern blots, this clone hybridized with a single 6.3 kb messenger ribonucleic acid (mRNA). In immunoblots of mouse brain extracts, antibodies raised in rabbits against the fusion protein encoded by it stained two glycoproteins of 135 and 90 kD, which we designated F3.135 and F3.90. In the developing mouse cerebellum, F3 antigenic sites were found predominantly on parallel fibers and on postmitotic neurons. In fetal brain cell cultures, F3 antigen was detected at the surface of cells with neuronal morphology, but the antibodies also stained some non-neuronal cells in a pattern characteristic of matrix components. Because all proteins carrying the L2/HNK-1 epitope identified so far have a role in cell adhesion, it can be anticipated that the F3 surface proteins also are involved in cell-interaction phenomena.

Animals↗

PubMed1986

Cloning, expression, and nucleotide sequence of livR, the repressor for high-affinity branched-chain amino acid transport in Escherichia coli.

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggest that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.

Amino Acid Sequence↗

PubMed1999

S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases.

S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) was partially purified from petals of the annual California plant Clarkia breweri. SAMT catalyzes the formation of methylsalicylate, an important floral scent compound in C. breweri, from salicylic acid and S-adenosyl-L-methionine (SAM). The native enzyme is a dimer with a subunit molecular weight of 40.3 kDa, and it has a Km for salicylic acid of 24 microM and a Km for SAM of 9 microM. A cDNA encoding SAMT was isolated from a C. breweri cDNA library prepared from floral mRNA. The sequence of the protein encoded by SAMT cDNA shows no significant sequence similarity to any protein in the data bank whose biochemical function is known. It does show significant sequence similarity (20-40% identity) to proteins encoded by at least seven Arabidopsis thaliana genes whose sequences have recently been determined in large-scale sequencing projects. The C. breweri SAMT cDNA was expressed in E. coli and the bacterial cells synthesized a functional SAMT protein with properties nearly identical to those of the plant-purified enzyme.

Amino Acid Sequence↗

PubMed2001

Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.

Amino Acid Sequence↗

PubMed1993

Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family.

Betacellulin (BTC) is a novel growth factor purified from the conditioned media of mouse pancreatic beta tumor cells and has been found to be a new member of the epidermal growth factor (EGF) family. The cDNA encoding human BTC has been cloned from a cDNA library prepared from human breast adenocarcinoma cell line MCF-7. The nucleotide sequence encodes a polypeptide which consists of 178 amino acid residues including a putative signal sequence, indicating that the structural organization of human BTC is similar to that of mouse BTC. The amino acid sequence of the human BTC precursor protein exhibits 79% similarity with that of the mouse precursor protein. The BTC gene was found to be expressed in several mouse tissues including kidney and liver as well as in a mouse beta tumor cell line and MCF-7 cells, suggesting that BTC might play a physiological role in normal tissues.

Amino Acid Sequence↗

PubMed1993

Cloning of a variant epidermal growth factor receptor.

The sequences encoding the full-length epidermal growth factor receptor (EGFR) were cloned from a cDNA library prepared from T3M4 cells. A transition (G to A) was identified at codon 497 of EGFR cDNA, resulting in the substitution of a lysine for an arginine. The same substitution was identified by sequencing cDNA derived from 3 of 7 additional human pancreatic cancer cell lines, one endometrial cancer cell line, and one lung cancer cell line, but not in A431 cells. Variant EGFR was always co-expressed with wild-type EGFR. Both sequences were present in genomic DNA from two cell lines expressing the variant receptor and in DNA from normal (3 of 7 individuals) human lymphocytes. These findings indicate that there are two alleles in this region of the EGFR gene, and that expression of variant EGFR is a common occurrence in normal and cancerous cells.

Base Sequence↗

PubMed1993

HuMig: a new human member of the chemokine family of cytokines.

Characterization of lymphokine-activated genes in mouse macrophages led to the identification previously of Mumig, an interferon-gamma-inducible murine gene that encodes a member of the chemokine family of cytokines. The Mumig cDNA probe was used to screen a cDNA library prepared from cultures of the THP-1 human monocytic cell line that had been treated with interferon-gamma. This led to the identification of Humig, a new human member of the chemokine gene family. Humig is induced in THP-1 cells and in peripheral blood mononuclear cells by interferon-gamma but not by interferon-alpha or by lipopolysaccharide. Analysis of mouse and human genomic DNAs suggested that the Mumig and Humig genes are true mouse-human homologues. The Humig mRNA encodes a predicted secreted HuMig protein of 103 residues, M(r) 11,725.

Amino Acid Sequence↗

PubMed1993

Cloning of cDNA encoding rat phospholipase C-beta 4, a new member of the phospholipase C.

Phospholipase C-beta 4(PLC-beta 4), a new member of phospholipase C isozyme, was purified from bovine cerebellum. The cDNA encoding rat PLC-beta 4 has been cloned from a cDNA library prepared from rat brain. The predicted open reading frame encodes a protein of 1,176 amino acids with a calculated molecular weight of 134,552. The deduced amino acid sequence exhibits 39, 36, and 36% identity with the sequences of rat PLC-beta 1, human PLC-beta 2, and rat PLC-beta 3, respectively. The amino acid sequence of PLC-beta 4, especially, shows higher identity (50%) with norpA PLC sequence from Drosophila melanogaster than those of other PLC-beta subtypes, suggesting that the PLC-beta 4 might be a mammalian PLC equivalent of norpA PLC implicated in photosignal transduction in Drosophila.

Amino Acid Sequence↗

PubMed1994

Isolation and structure of cat superfast myosin light chain-2 cDNA and evidence for the identity of its human homologue.

A full-length cDNA clone coding for superfast myosin light chain-2 (MyLC2) was isolated from an expression cDNA library prepared from cat masseter muscle and was fully characterized. The deduced amino acid sequence shares 58% overall identity with limb fast MyLC2, whereas homologies of the latter among higher vertebrates show 90% identity, indicating that superfast MyLC2 has diverged considerably from limb fast MyLC2 during evolution. Superfast MyLC2 cDNA has 89% nucleotide homology and 93% amino acid homology with a published novel human MyLC2 (MYL5), suggesting that MYL5 is a human homologue of the cat superfast MyLC2. Hybridization of a superfast MyLC2 isoform-specific probe reveals that expression of superfast MyLC2 in cat is confined to jaw-closing muscles. In conclusion, the present paper describes for the first time the cloning of a superfast myosin light chain coding sequence.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and expression of the gene encoding human angiotensin II type 2 receptor.

The gene of human angiotensin II type 2 (AT2) receptor was isolated from a genomic DNA library prepared from human placenta. The coding region of the human AT2 receptor gene was contained in a single exon coding segment of the gene indicating an intronless structure of the coding region. The amino acid sequence of human AT2 receptor deduced from its nucleotide sequence has 363 amino acids and shows a high degree of sequence identity to rat and mouse receptor sequences. Specific binding of [125I]Sar1Ile8-angiotensin II was demonstrated in COS-7 cells transfected with a plasmid containing the human AT2 sequence. Scatchard analysis and ligand displacement profile were typical of the AT2 receptor. Reverse transcription-polymerase chain reaction analysis showed that AT2 receptor mRNA was expressed in adult uterus and pheochromocytoma.

Amino Acid Sequence↗

PubMed1994

Calcium modulates the cyclin D1 expression in a rat parathyroid cell line.

We have used a rat epithelial parathyroid cell line (PT-r) to study the expression and regulation of D-type cyclins. In PT-r cells the cyclin D1 gene is the most abundantly expressed, being transcribed in at least two mRNAs whose levels oscillate during the cell cycle. We also screened a cDNA library prepared from PT-r cells with the human cyclin D1 probe and isolated its rat homologue. Cyclin D2 and D3 mRNAs are both represented in PT-r cells but the former one is only barely detectable. Moreover, the oscillation of cyclin D3 transcript is slightly delayed when compared to cyclin D1 and D2. Since extracellular calcium inhibits parathyroid cell proliferation, we looked for the effect of the ion on the expression of cyclin D genes in PT-r cells. Increasing amounts of calcium in the incubation medium reduced the expression of rat cyclin D1 and D2. The effect appears to be cell-specific and probably mediated through the inhibition of mitogenic signalling pathways.

Amino Acid Sequence↗

PubMed1996

Members of the MAZ family: a novel cDNA clone for MAZ from human pancreatic islet cells.

A human recombinant cDNA clone that encoded a zinc-finger protein (Myc-associated zinc-finger protein of human islet; MAZi) was cloned by screening a cDNA library prepared from human pancreatic islet cells. The encoded protein showed a high degree of homology to the Myc-associated zinc-finger protein MAZ (ZF87 or Pur-1). However, differences between the cDNAs for MAZi and MAZ were found in the length of the encoded polyalanine stretch and in the sequence of the 5'-end leader. MAZi transcripts were significantly more abundant in rat pancreatic islet carcinoma tissue than in normal rat islet cells. Moreover, MAZi protein bound specifically to the pyrimidine-rich strand of the CT-element of the c-myc gene in vitro and strongly induced the expression of chloramphenicol acetyltransferase (CAT) from a c-myc promoter/ CAT reporter construct in human pancreatic cells. Our results suggest that a distinct member of the MAZ family is expressed in human islet cells and enhances the transcriptional activity of the c-myc gene.

Amino Acid Sequence↗

PubMed1997

Sequence of a cDNA clone encoding a novel somatolactin in goldfish, Carassius auratus.

As a first step towards the development of a sensitive ribonuclease protection assay to study the regulation of somatolactin (SL) mRNA expression in pituitary cells of goldfish, we have isolated a complementary DNA (cDNA) clone encoding precursor sequence of SL from a cDNA library prepared from goldfish pituitary poly(A)+ RNA. The 843-bp goldfish SL (gfSL) cDNA has an open reading frame of 693 nucleotides with two possible start codons of AUG. Amino acid sequence alignment revealed that gfSL has the characteristics of four conserved domains (A, B, C and D) common to all SLs with the domain B being the most conserved region among all the characterized SLs. Similar to other teleost SLs, this gfSL is similarly related but clearly distinct from growth hormone and prolactin of goldfish and other teleosts. However, unlike most other known teleost SLs which have more than 70% amino acid sequence identity to each other, the overall amino acid sequence identity of this novel gfSL with other previously characterized SLs ranges from only 36% to 51%. Moreover, this gfSL contains only six cysteine residues, rather than seven in most other SLs, in conserved positions. Northern blot analysis revealed a single gfSL mRNA transcript of approximately 1 kb in the pituitaries of both sexually regressed and maturing male and female goldfish.

Amino Acid Sequence↗

PubMed1997

Cloning and sequencing of aspartate aminotransferase from Thermus aquaticus YT1.

A 39-base oligonucleotide "guessmer" probe, based on partial N-terminal sequence analysis of the aspartate aminotransferase purified from Thermus aquaticus strain YT1, was used to screen a genomic library prepared from T. aquaticus DNA. A 1842 bp DNA fragment was isolated that proved to contain the coding sequence for the aspartate aminotransferase. The gene is 1152 bases long and codes for a protein of 383 amino acid residues. The amino acid sequence obtained showed 88.7%, 45.1% and 32.9% identity of sequence with those of thermostable aspartate aminotransferases from T. thermophilus, Bacillus YM2, and Sulfolobus solfataricus, respectively. It showed 39.1% identity with one of the gene products tentatively identified as aspartate aminotransferase from the methanogenic archaebacterium Methanococcus jannaschii. Neither the amino acid compositions nor the aligned amino acid sequences provides any obvious clue as to the origin of thermal stability in this group of enzymes.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and sequence analysis of chicken type I deiodinase cDNA: expression in normal and dwarf broiler chickens.

A cDNA encoding the chicken type I iodothyronine deiodinase (cDI-1) was isolated and sequenced from a cDNA library prepared from ConA-activated chicken splenic T-lymphocytes. The coding region of cDI-1 cDNA is composed of 738 basepairs (bp) which encodes a 246 amino acid protein. The predicted amino acid sequence of cDI-1 indicates only 60% identity to several mammalian type I deiodinases. The cDI-1 cDNA contains a codon for a highly conserved selenocysteine residue (Cys124). Northern blot analysis of total RNA prepared from different tissues of a 3-week-old broiler chicken shows a single transcript (2 kb) in liver and kidney. The abundance of hepatic cDI-1 transcripts in growth hormone receptor (GHR)-deficient dwarf chicken was similar to normal chickens despite lower levels of plasma T3 (37% lower) and elevated levels of T4 (21% higher) in dwarf chickens. This finding suggests that regulation of hepatic cDI-1 mRNA is GH-independent in the post-hatch chicken.

Amino Acid Sequence↗