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PubMed1994

Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents.

Alcohol Oxidoreductases↗

PubMed1989

Molecular structure and expression of the murine lymphocyte low-affinity receptor for IgE (Fc epsilon RII).

The cDNA encoding the murine low-affinity receptor for IgE (Fc epsilon RII) has been isolated from a cDNA library prepared from B cells activated with lipopolysaccharide and interleukin 4. It encodes a 37-kDa protein of 331 amino acids with two potential N-linked glycosylation sites. Analogous to its human counterpart, there is no signal sequence and the putative transmembrane region is close to the amino terminus, indicating an inverse membrane orientation with the carboxyl terminus at the cell exterior. The predicted murine Fc epsilon RII amino acid sequence demonstrates a 57% identity with its human counterpart. The murine sequence has an additional internal repeat motif of 21 amino acids giving four repeats as compared to three in the human sequence. Furthermore, the murine Fc epsilon RII is truncated at the carboxyl terminus and the Arg-Gly-Asp sequence, a common recognition site of integrin receptors, which is found in the reverse configuration in human Fc epsilon RII, is missing. B cells activated with interleukin 4 and lipopolysaccharide have an increased amount of Fc epsilon RII mRNA as compared with resting or lipopolysaccharide-stimulated B cells. Con A-activated normal T cells, the TH-2 cell line D10, as well as the macrophage cell line J774 have no detectable Fc epsilon RII mRNA. Expression analysis using transiently transfected COS cells revealed that recombinant murine Fc epsilon RII binds anti-Fc epsilon RII as well as mouse and rat IgE but does not bind human IgE or mouse IgG. Fc epsilon RII expressed in COS cells has a molecular mass of 45 kDa whereas the Fc epsilon RII from B-cell lines is a 49-kDa protein.

Amino Acid Sequence↗

PubMed1990

Characterization of a novel, human cytotoxic lymphocyte-specific serine protease cDNA clone (CSP-C).

A human cDNA clone encoding a novel serine protease, cytotoxic serine protease-C(CSP-C), has been isolated from a cDNA library prepared from recombinant interleukin-2 (IL-2)-activated lymphocytes of a patient with a large granular lymphoproliferative disorder. The clone has a 741-base pair open reading frame encoding a putative 246-amino acid protein. The protein sequence contains the catalytic charge relay system characteristic of a serine protease and the conserved N-terminal amino acid sequence of the mature cytotoxic lymphocyte serine proteases found in both mouse and human. The amino acid sequence of CSP-C has 71% identity with the previously reported cytotoxic serine protease-B(CSP-B)/human lymphocyte protease (HLP)/SECT and 57% identity with the granulocyte-specific serine protease cathepsin G. The homology with another lymphocyte-specific serine protease, human Hanukah factor (HF)/Granzyme A was 41%. The transcript is expressed in lymphocytes stimulated with IL-2 or IL-2 plus phytohemagglutinin (PHA). CSP-C is not expressed in B-lymphoblastoid cell lines or in the T-leukemia cell line MOLT4. The cDNA sequence suggests that the protein is expressed as a prepropeptide, as has been found in the other murine and human serine proteases of lymphocyte origin. It has recently been reported that human chromosome 14q11, in addition to containing the genes encoding cytotoxic serine protease B (CSP-B), cathepsin G, and the T-cell receptor alpha and delta genes, also includes an additional genomic DNA clone which cross-hybridized with CSP-B and cathepsin G, cathepsin-like gene-2 (CGL-2). It is likely that the CSP-C cDNA clone reported in this study corresponds to CGL-2.

Amino Acid Sequence↗

PubMed1991

Cloning of the gene encoding the human erythropoietin receptor.

The genomic and complementary DNAs of the human erythropoietin receptor (hEpo-R) have been isolated and characterized from a genomic placental library and from two cDNA libraries prepared from bone marrow and fetal liver. The five different partial cDNAs isolated were aberrant in the predicted reading frames as compared with the Epo-R protein sequence, because all retained insert sequences that may represent splicing intermediates (three clones), cloning artifact (one clone), or a new sequence at a splice junction (one clone) of the gene. The cDNAs were used to isolate several genomic clones encompassing the complete hEpo-R gene. This gene, which encodes a 508-amino acid polypeptide chain of predicted M(r) 55,000, is organized into eight exons spread over 6 kb of DNA and exhibited a high degree of sequence homology (81.6% in the coding region) and structural organization with its murine counterpart. Primer extension analysis indicated that the transcription initiation site is located 141 bp upstream of the initiation codon. Sequence homology 320 bp upstream of the cap site was significantly lower (60%) and diverged completely further upstream as compared with the murine gene. Similarly, the human and murine sequences were largely divergent downstream of the stop codon, indicating that a strong conservation during evolution was restricted to the coding sequence of the Epo-R protein. The 320-bp region upstream of the cap site does not contain the typical TATA or CAAT boxes present in many tissue-specific genes, but does include potential binding sites for the ubiquitous Sp1 and the erythroid-specific GATA-1 trans-activating factors. These boxes are well conserved in sequence and position relative to the cap site within the promoter region of the human and murine genes, but the CACCC boxes present in the murine gene are absent in the human gene.

Animals↗

PubMed1993

Structural and functional characterization of rev-like transcripts of equine infectious anemia virus.

Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.

Amino Acid Sequence↗

PubMed1991

Primary structure of the human laminin A chain. Limited expression in human tissues.

cDNA clones for the human laminin A chain were isolated from libraries prepared from human gestational choriocarcinoma cell line (JAR) RNA. They cover approx. 8 kb from the 5'-end of the 9.5 kb mRNA coding for this protein. Our clones contain 94 nucleotide residues for the 5'-end untranslated region and 7885 nucleotide residues of coding sequence. The complete human laminin A chain contains a 17-amino acid-residue signal peptide and a 3058-residue A chain proper. The human laminin A chain has a distinct domain structure with numerous internal cysteine-rich repeats. The large globular domain G has five repeats, which have several conserved glycine and cysteine residues. Furthermore the A chain contains 20 internal cysteine-rich repeats present in tandem arrays in three separate clusters (domains IIIa, IIIb and V). Domain I + II has a predicted continuous alpha-helical structure characterized by heptad repeats and three domains (IVa, IVb and VI) are predicted to contain a number of beta-sheets and coiled-coil structures. Northern-blot analysis was used to study the laminin A chain expression in the JAR cell line, full-term placenta and newborn-human tissues (kidney, spleen, lung, heart muscle, psoas muscle and diaphragm muscle). The expression was detectable in newborn-human kidney and JAR cell line only. The overall amino acid sequence identity between human and mouse is 76%. The human chain has only one Arg-Gly-Asp (RGD) sequence, which is located in the long arm within domain G, whereas the single RGD sequence in the mouse chain is located in the short arm in domain IIIb. The degree of identity between the human laminin A chain sequence and the sequence available for merosin [Ehrig, Leivo, Argraves, Ruoslahti & Engvall (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268] is about 41% and when conservative substitutions are included the degree of similarity is 54%.

Amino Acid Sequence↗

PubMed1995

A tetraspan membrane glycoprotein produced in the human intestinal epithelium and liver that can regulate cell density-dependent proliferation.

The human cell line HT-29 provides a model system for studying regulation of proliferation and differentiation in intestinal epithelial cell lineages: (i) HT-29 cells cultured in glucose resemble undifferentiated multipotent transit cells located in the lower half of intestinal crypts; (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt; (iii) nonproliferating, confluent HT-29-inosine cells have features of differentiated enterocytes and goblet cells that overlie small intestinal villi. A cDNA library prepared from HT-29-inosine cells was screened with a series of subtracted cDNA probes to identify proteins that regulate proliferation/differentiation along the crypt-villus axis. A cDNA was recovered that encodes a 202-amino acid protein with four predicted membrane spanning domains and two potential sites for N-linked glycosylation. Levels of this new member of the superfamily of tetraspan membrane proteins (TMPs) increase dramatically as nondividing epithelial cells exit the proliferative compartment of the crypt-villus unit and migrate onto the villus. The protein is also produced in nondividing hepatocytes that have the greatest proliferative potential within liver acini. Three sets of observations indicate that in the appropriate cellular context, intestinal and liver (il)-TMP can mediate density-associated inhibition of proliferation. (i) Accumulation of il-TMP glycoforms precedes terminal differentiation of HT-29-inosine cells and occurs as they undergo density-dependent cessation of growth. il-TMP levels are lower and glycosylation less extensive in HT-29-glucose cells, which do not undergo growth arrest at confluence. (ii) HeLa cells normally do not produce il-TMP. Forced expression of il-TMP inhibits proliferation as cells approach confluence. The extent of il-TMP glycosylation in the transfected cells is similar to that observed in HT-29-inosine cells and greater than in HT-29-glucose cells. (iii) SW480 cells are derived from a human colon adenocarcinoma and do not express il-TMP. Like nontransfected HeLa cells, they do not stop dividing at confluence, whether grown in medium containing glucose or inosine. Expression of il-TMP has no effect on the growth properties of SW480 cells. The extent of il-TMP glycosylation in SW480-glucose cells is similar to that noted in HT-29-glucose cells, lending further support to the notion that il-TMP's activity is related to its state of N-glycosylation.

Adult↗

PubMed1997

Regulation of two loblolly pine (Pinus taeda L.) isocitrate lyase genes in megagametophytes of mature and stratified seeds and during postgerminative growth.

Two full-length cDNAs encoding the glyoxysomal enzyme isocitrate lyase (ICL) were isolated from a lambda ZAP cDNA library prepared from megagametophyte mRNAs extracted from seeds imbibed at 30 degrees C for 8 days. The cDNAs, designated Ptbs ICL 8 and Ptbs ICL 12, have open reading frames of 1740 and 1719 bp, with deduced amino acid sequences of 580 and 573 residues, respectively. The predicted amino acid sequences of Ptbs ICL 8 and Ptbs ICL 12 exhibit a 79% identity with each other, and have a greater than 75% identity with ICLs from various angiosperm species. The C-termini of Ptbs ICL 8 and Ptbs ICL 12 terminate with the tripeptide Ser-Arg-Met and Ala-Arg-Met, respectively, both being conserved variants of the type 1 peroxisomal targeting signal. RNA blot and slot analysis revealed that Ptbs ICL 8 and Ptbs ICL 12 mRNAs were present at low levels in the megagametophyte of the mature and stratified seeds, and that the level of both transcripts increased markedly upon seed germination. Protein blot analysis indicated that the steady-state level of ICL was low in the mature and stratified seed, then increased rapidly upon seed germination, peaking at around 8-10 days after imbibition (DAI). Changes in the level of ICL activity in cell-free extracts was similar to the steady-state protein content with the exception that ICL activity was not detected in megagametophyte extracts of mature or stratified seeds. From 10-12 DAI when the megagametophyte tissue senesced, ICL activity decreased rapidly to near undetectable levels. In contrast, steady-state levels of ICL protein and mRNA remained relatively constant during megagametophyte senescence. In vivo synthesis of ICL protein was measured to shed light on these differences. ICL immunoselected from [(35)S]-methionine labelled proteins indicated that ICL was synthesized at very low levels during megagametophyte senescence. Together, the results show that loblolly pine ICL gene expression is complex. While temporal regulation appears to be primarily transcriptional, it also involves a number of post-transcriptional processes including at least one translational and/or post-translational mechanism.

Amino Acid Sequence↗

PubMed1989

Two arylamine N-acetyltransferases from chicken pineal gland as identified by cDNA cloning.

A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.

Acetyltransferases↗

PubMed1998

Secretion of neu differentiation factor-like polypeptides by cph-transformed fibroblasts: cloning and characterization of Syrian hamster neuregulin cDNAs.

We reported previously that expression of the cph oncogene in Syrian hamster and mouse cells leads to the secretion of a polypeptide factor or factors structurally and functionally related to neu differentiation factor (NDF) and the establishment of an autocrine loop mediated through the simultaneous overexpression of the erbB4 receptor. To identify the nature of this factor and to characterize its role in the neoplastic conversion of Syrian hamster embryo cells, we cloned cDNAs hybridizing to rat NDF-derived probes by screening a library prepared from neoplastic 81C39 cells, which harbor an activated cph oncogene and secrete active NDF-like polypeptides. Sequence analysis of the isolated clones revealed a high level of homology between the hamster neuregulin and its rat and human counterparts and the existence of various neuregulin cDNA variants in Syrian hamster cells, presumably originating from alternatively spliced RNA species. Interestingly, some of these neuregulin cDNAs were longer (up to 5.5 kb) than those isolated before from any other system, suggesting that the Syrian hamster neuregulin precursor mRNA accumulates at greater concentrations than in other species. We also detected different hamster neuregulin protein isoforms by using in vitro transcription-translation analyses. Peptide sequence analysis identified the major NDF-like polypeptide secreted by 81C39 cells as an alpha2b-neuregulin. Northern blot analyses with the cloned cDNA inserts showed that neuregulin overexpression was commonly associated with the neoplastic conversion of chemically initiated hamster embryo fibroblasts. This, along with the detection of elevated erbB2- or erbB4-specific transcripts in most (six of eight) neoplastic cell lines tested, supports the notion that autocrine neuregulin signaling plays an important role in maintaining the transformed phenotype by providing a growth advantage to cph-transformed cells.

Amino Acid Sequence↗

PubMed1997

New member of the Snf1/AMPK kinase family, Melk, is expressed in the mouse egg and preimplantation embryo.

The initial phase of mammalian preimplantation development is directed by stored maternal mRNAs and their encoded proteins, yet most of the molecules controlling this process have not been described. We have used differential display analysis of cDNA libraries prepared from unfertilized eggs and preimplantation embryos to isolate three maternal cDNAs that represent novel genes exhibiting different patterns of expression during this developmental period. One of these, Melk, encodes a protein with a kinase catalytic domain and a leucine zipper motif, a new member of the Snf1/AMPK family of kinases. This gene product may play a role in the signal transduction events in the egg and early embryo.

Amino Acid Sequence↗

PubMed1996

POU homeodomain genes and myogenesis.

We show that members of the POU homeodomain family are among the transcription factors expressed in developing mouse skeletal muscle. From a cDNA library prepared from fetal muscle mRNA, we cloned a cDNA identical to that of Brn-4, a POU class II gene previously cloned from neural tissues. In limb muscle, we found that Brn-4 mRNA expression was highest at embryonic days 15-18, declined-after birth, and was undetectable in adults. The mRNAs of two additional POU genes, Emb (POU class VI) and Oct-1 (POU class II), were also expressed in developing muscle and, unlike Brn-4, continued to be expressed in postnatal and adult muscles. In skeletal muscle, expression of Brn-4 is myogenin-dependent, because muscles from myogenin-deficient fetuses contained much less Brn-4 mRNA than muscles from myogenin-expressing littermates. In contrast, expression of Emb was the same in the presence or absence of myogenin. The distinct pattern of Brn-4 mRNA expression and its dependence on a myogenic regulatory factor suggest that Brn-4 is part of the network of interacting transcription factors that control muscle-specific gene expression during mammalian myogenesis.

3T3 Cells↗

PubMed2002

Macrocyclic hairpin mimetics of the cationic antimicrobial peptide protegrin I: a new family of broad-spectrum antibiotics.

The problems associated with increasing antibiotic resistance have stimulated great interest in newly discovered families of naturally occurring cationic antimicrobial peptides. These include protegrin, tachyplesin, and RTD-1, which adopt beta-hairpin-like structures. We report here an approach to novel peptidomimetics based on these natural products. The mimetics were designed by transplanting the cationic and hydrophobic residues onto a beta-hairpin-inducing template, either a D-Pro-L-Pro dipeptide or a xanthene derivative. The mimetics have good antimicrobial activity against Gram-positive and Gram-negative bacteria (minimal inhibitory concentration approximately 6-25 microgram mL(-1)). Analogues with improved selectivity for microbial rather than red blood cells (1 % hemolysis at 100 microgram mL(-1)) were identified from a small library prepared by parallel synthesis. Thus, it is possible to separate the antimicrobial and hemolytic activities in this class of mimetics. NMR studies on one mimetic revealed a largely unordered structure in water, but a transition to a regular beta-hairpin backbone conformation in the presence of dodecylphosphocholine micelles. This family of mimetics may provide a starting point for the optimization of antimicrobial agents of potential clinical value in the fight against multiple-drug-resistant microorganisms.

Anti-Bacterial Agents↗

PubMed2000

DC-STAMP, a novel multimembrane-spanning molecule preferentially expressed by dendritic cells.

Dendritic cells (DC) are unique in their ability to present antigen to naive T cells, and therefore play a central role in the initiation of immune responses. Characterization of DC-specific genes may help to unravel the mechanism underlying their potent antigen presenting capacity. Here we describe the identification of a novel transcript, isolated by random sequencing of a cDNA library prepared from monocyte-derived DC, which we termed DC-specific transmembrane protein (DC-STAMP). DC-STAMP is specifically expressed by DC, and not in a panel of other leukocytes or non-hematopoietic cells. Interestingly, DC-STAMP was also detected in activated but not resting blood DC. The DC-STAMP transcript encodes a 470-amino acid protein containing seven putative transmembrane domains. Expression of a DC-STAMP-GFP fusion protein in 293 cells indicates that DC-STAMP is expressed at the cell surface, and has an intracellular C terminus. Surprisingly, no sequence homology was found with any other protein or multimembrane-spanning receptor. Therefore, we propose that DC-STAMP is a novel DC-specific multimembrane-spanning protein, representing a new group of transmembrane proteins.

Amino Acid Sequence↗

PubMed2005

Discovery of powerful uranyl ligands from efficient synthesis and screening.

New tripodal gem-(bis-phosphonates) uranophiles were discovered by a screening method that allowed for the selection of ligands with strong uranyl-binding properties in a convenient microtiter-plate format. The method is based on competitive uranium binding by using Sulfochlorophenol S as chromogenic chelate. This dye compound was found to present high uranyl complexation properties and allowed to highlight ligands presenting association constants for UO(2+)(2) up to 10(18) at pH 7.4 and 10(20) at pH 9. A collection of 40 known ligands including polycarboxylate, hydroxamate, catecholate, hydroxypyridonate and hydroxyquinoline derivatives was tested. Also screened was a combinatorial library prepared from seven amine scaffolds and eight acrylates bearing diverse chelating moieties. Among these 96 tested candidates, a tripod derivative bearing gem-bis-phosphonates moieties was found to present the highest complexation properties over a wide range of pH and was further studied.

Hydrogen-Ion Concentration↗

PubMed2006

Microwave-assisted Suzuki coupling reactions with an encapsulated palladium catalyst for batch and continuous-flow transformations.

This article describes the design, optimisation and development of a Suzuki cross-coupling protocol mediated by an efficient palladium-encapsulated catalyst (Pd EnCat) under microwave irradiation. The methodology has been used in both batch mode for classical library preparation and in continuous-flow applications furnishing multigram quantities of material. Described is a method that uses direct focused microwave heating whilst applying an external cooling source. This enables a lower than normal bulk temperature to be maintained throughout the reaction period leading to significant improvements in the overall yield and purity of the reaction products. Additional aspects of this novel heating protocol are discussed in relation to the prolonged lifetime and enhanced reactivity of the immobilised catalyst system.

Catalysis↗

PubMed1993

Sea urchin maternal mRNA classes with distinct development regulation.

Previous studies of newly synthesized proteins during early development in sea urchins have revealed several different patterns of synthesis that can be used to predict the existence of mRNA classes with distinct regulatory controls. We have identified clones for abundant maternal mRNAs that are actively translated during early development by screening a cDNA library prepared from polysomal poly(A)+RNA isolated from 2-cell stage (2-hour) Strongylocentrotus purpuratus embryos. Probes prepared from these cDNA clones and several previously characterized maternal mRNA cDNAs were used to compare relative levels of individual mRNAs in eggs and embryos and their translational status at various developmental stages. These abundant mRNAs can be classified into two major groups which we have termed cleavage stage-specific (CSS) and post cleavage stage (PCS) mRNAs. The relative levels of the CSS mRNAs are highest during the rapid cleavage stage and decrease dramatically at the blastula stage (12-hours). In contrast, PCS mRNAs are present at relatively low levels during the rapid cleavage stage and then increase at the blastula stage. Polysome partition profiles reveal that CSS mRNAs are translated more efficiently than PCS mRNAs in the unfertilized egg, at fertilization, and during the cleavage stages. Following the blastula stage, some CSS transcripts move out of polysomes and accumulate as untranslated RNAs, while newly transcribed PCS mRNAs are recruited into polysomes. These data suggest that the rapid cell cycles following fertilization require high levels of specific cleavage stage proteins, and the synthesis of these proteins occurs preferentially over PCS mRNAs.

Animals↗

PubMed1988

cDNA cloning of murine interleukin-HP1: homology with human interleukin 6.

Interleukin-HP1 (HP1) is a murine T cell-derived lymphokine, originally described as a growth factor for B cell hybridomas and plasmacytomas, that was recently shown to stimulate growth and differentiation of normal B and T lymphocytes. Here, we describe a cDNA for HP1 that was isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody. The cDNA, which hybridizes with a mRNA of approximately 1300 bp, encodes a polypeptide consisting of 211 amino acids with a typical signal sequence of 24 residues followed by 187 amino acids, which form the mature protein (Mr = 21,710). No N-glycosylation site but several potential O-glycosylation sites were identified in the predicted sequence. Comparison of the cDNA sequence of HP1 with that of human interleukin 6 disclosed a homology of 65% at the DNA level and of 42% at the protein level with a maximum of 57% for the segment spanning residues 42-102 of mature HP1. Considering the functional homology that was previously established between these two proteins, we therefore propose that HP1 be renamed murine interleukin 6.

Amino Acid Sequence↗