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PubMed1982

The nucleotide sequence of mRNA for the Mr 19 000 glycoprotein from early gene block III of adenovirus 2.

The cytoplasmic poly(A)RNA from early stages of infection of HeLa cells by adenovirus 2 (Ad2), was used to synthesize cDNA. The resulting cDNA segments were inserted at the PstI site of the plasmid pBR322 after dG/dC tailing. The clones containing sequences corresponding to early region III transcripts were identified by hybridization against the SmaI-C fragment (76.5 to 91.9 map units) of the Ad2 genome. The clone pE22 which contained a copy of the mRNA coding for the Mr 19 000 glycoprotein was characterized in detail. A comparison with the genomic sequences allowed the identification of the splice between the x and y leaders. The nucleotide sequences CCGGTG and CAGTTT were found at the donor and acceptor sites of the splice junction, respectively. The coding region consisted of a continuous stretch of 159 amino acids with a hydrophobic N-terminus and two possible glycosylation sites. The triplet ATG was encountered twice in phase 3 before the actual site of initiation of translation, which was in phase 2. The 3'-untranslated region was 500 nucleotides long and contained an open, translational reading frame for a Mr 11 000 protein, following a potential initiator ATG. The sequence ATTAAA was observed 17 bp before the poly(A) tail.

Adenoviruses, Human↗

PubMed2001

Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length.

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.

5' Untranslated Regions↗

PubMed1998

AUUUA sequences direct mRNA deadenylation uncoupled from decay during Xenopus early development.

To study the regulation of AUUUA-mediated RNA deadenylation and destabilization during Xenopus early development, we microinjected chimeric mRNAs containing Xenopus or mammalian 3' untranslated region (3'-UTR) sequences into Xenopus oocytes, mature eggs, or fertilized embryos. We found that the AU-rich elements (ARE) of Xenopus c-myc II and the human granulocyte-macrophage colony-stimulating factor gene (GMCSF) both direct deadenylation of chimeric mRNAs in an AUUUA-dependent manner. In the case of the Xenopus c-myc II ARE, mutation of a single AUUUA within an absolutely conserved 11-nucleotide region in c-myc 3'-UTRs prevents ARE-mediated deadenylation. AUUUA-specific deadenylation appears to be developmentally regulated: low deadenylation activity is observed in the oocyte, whereas rapid deadenylation occurs following egg activation or fertilization. Deadenylation results in the accumulation of stable deadenylated RNAs that become degraded only following mid-blastula transition. We conclude that ARE-mediated mRNA deadenylation can be uncoupled from ARE-mediated mRNA decay and that AUUUAs directly signal deadenylation during Xenopus early development.

3' Untranslated Regions↗

PubMedDate not supplied

In situ cellular analysis of alpha-fetoprotein gene expression in regenerating rat liver after partial hepatectomy.

Cellular analysis of hepatic alpha-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few alpha-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of alpha-fetoprotein secretion, 10 to 20% alpha-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2,6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of alpha-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in alpha-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of alpha-fetoprotein mRNA sequences and a late cytoplasmic accumulation of alpha-fetoprotein mRNA molecules.

Animals↗

PubMed1984

Interaction of L and NS proteins of vesicular stomatitis virus with its template ribonucleoprotein during RNA synthesis in vitro.

Soluble transcriptase containing the L and the NS proteins was isolated from purified vesicular stomatitis virus and its binding with the template ribonucleoprotein containing the N protein-RNA complex was studied with respect to its ability to initiate and synthesize RNA in vitro. By using u.v.-irradiated template reconstituted with soluble transcriptase, it was shown that the synthesis of leader RNA and other small initiated mRNA sequences continued while full-length mRNA synthesis decreased by 90%. In the presence of ATP and CTP, the reconstituted complex synthesized polyphosphorylated oligonucleotides which include AC, AAC and AACA which represent 5'-terminal sequences transcribed from the leader template and genes coding for mRNAs. In the presence of arabinosyl ATP, an inhibitor of RNA synthesis in vitro, the synthesis of leader RNA was found to be inhibited considerably more than other small initiated mRNA sequences. Reconstitution of RNA synthesis with soluble transcriptase and template in the presence of viral matrix (M) protein at low ionic condition resulted in virtual cessation of leader RNA synthesis, although the synthesis of small initiated N mRNA, 11 to 14 bases, continued. These results suggest that transcriptase can bind at multiple sites on the genome template and initiate RNA chains.

DNA-Directed RNA Polymerases↗

PubMed2001

CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist.

1. Calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) form the basis of all the receptors in this family of peptides. 2. Using reverse transcriptase - polymerase chain reaction the presence of mRNA sequences encoding the CRLR, RAMP1 and RAMP2 were demonstrated in porcine left anterior descending (LAD) coronary arteries, whereas porcine calcitonin (CT) receptor mRNA was not present. The partial porcine mRNA sequences shared 82 - 92% nucleotide identity with human sequences. 3. The human peptides alphaCGRP, betaCGRP, AM and amylin induced relaxation with pEC(50) values of 8.1, 8.1, 6.7 and 6.1 M respectively. 4. The antagonistic properties of a novel non-peptide CGRP antagonist 'Compound 1' (WO98/11128), betaCGRP(8 - 37) and the proposed AM receptor antagonist AM(22 - 52) were compared to the well-known CGRP(1) receptor antagonist alphaCGRP(8 - 37). 5. The alphaCGRP(8 - 37) and betaCGRP(8 - 37) induced concentration-dependent (10(-7) - 10(-5) M) rightward shift of both the alphaCGRP and betaCGRP concentration-response curves. betaCGRP(8 - 37) (10(-6) M) had the same effect as alphaCGRP(8 - 37) (10(-6) M), but with less potent rightward shift of the concentration-response curves for alphaCGRP, AM and amylin. 6. Preincubation with 'Compound 1' (10(-7) - 10(-5) M) and AM(22 - 52) (10(-6) M) had no significant antagonistic effect. 7. In conclusion, the building blocks forming CGRP and AM receptors were present in the porcine LAD, whereas those of the amylin receptor were not. alphaCGRP, betaCGRP, AM and amylin mediated vasorelaxation via the CGRP receptors. No functional response was detected to adrenomedullin via the adrenomedullin receptor.

Adrenomedullin↗

PubMed1999

Unique 5'-end of a Na(+)-K(+)-2Cl- cotransporter-like mRNA expressed in rat skeletal muscle.

Functional evidence presented by others indicates that rat slow-twitch skeletal muscle lacks typical Na(+)-K(+)-2Cl- cotransporter activity, as determined by loop diuretic-sensitive potassium transport. This report presents a unique 5' mRNA sequence of a Na(+)-K(+)-2Cl- cotransporter-like molecule expressed in the rat soleus muscle and the deduced N-terminus of the protein. In addition to its unique 5' mRNA sequence, the coding region of the N-terminus is quite short compared with other known Na(+)-K(+)-2Cl- cotransporters. Nonetheless, the mRNA possesses conserved cotransporter-like membrane spanning domains, though one domains corresponding to a reported exon is divergent. Therefore, it appears that skeletal muscle does express a Na(+)-K(+)-2Cl- cotransporter-like mRNA that may code for a protein with atypical Na(+)-K(+)-2Cl- cotransporter properties.

Amino Acid Sequence↗

PubMed1987

Superinduction of the human gene encoding immune interferon.

Mitogen-induced interferon-gamma (IFN-gamma) gene expression was analyzed in human tonsil cells by titration of IFN-gamma activity and by quantitation of IFN-gamma mRNA. Expression of the IFN-gamma gene can be superinduced extensively by two distinct methods: exposure to various inhibitors of translation, or to low doses of gamma-irradiation. gamma-Irradiated cells produce, after exposure to cycloheximide, up to 12-fold greater amounts of IFN-gamma activity. Within as little as 4 h after the addition of translation inhibitors, IFN-gamma mRNA levels rise 3- to 5-fold. Superinduction acts to increase the size of the wave of IFN-gamma mRNA. Primary transcription of the IFN-gamma gene does not increase in cells superinduced by cycloheximide, nor can superinduction be explained by stabilization of IFN-gamma mRNA sequences. These findings show that, during normal induction, a labile protein acts post-transcriptionally to repress the accumulation of mature IFN-gamma mRNA sequences. The superinductive effects of cycloheximide and gamma-irradiation on levels of IFN-gamma are additive, suggesting that they affect different aspects of IFN-gamma gene expression. Superinduction by gamma-irradiation also has a post-transcriptional basis and is consistent with the possibility that expression of the IFN-gamma gene is normally controlled by the action of suppressor T cells. Even though the genes for human IFN-gamma and for interleukin-2 are both superinducible, a striking difference in the regulation of expression of these lymphokine genes is observed. Superinduction of IFN-gamma mRNA is not due to superinduction of interleukin-2.

Cells, Cultured↗

PubMed2003

ProSplicer: a database of putative alternative splicing information derived from protein, mRNA and expressed sequence tag sequence data.

ProSplicer is a database of putative alternative splicing information derived from the alignment of proteins, mRNA sequences and expressed sequence tags (ESTs) against human genomic DNA sequences. Proteins, mRNA and ESTs provide valuable evidence that can reveal splice variants of genes. The alternative splicing information in the database can help users investigate the alternative splicing and tissue-specific expression of genes.

Alternative Splicing↗

PubMed1981

Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei.

Crude microsomes from lactating rabbit mammary gland were incubated with prolactin. The incubation mixture was centrifuged and the supernatant was incubated with isolated mammary cell nuclei from lactating rabbits treated for 4 days by bromocryptin to antagonize prolactin and to deinduce casein gene transcription. Nuclei were incubated with HgCTP, and the newly synthesized mercurated RNA was isolated on SH-Sepharose columns. The content of beta-casein mRNA sequences in the fraction eluted with 2-mercaptoethanol was estimated with a [(3)H]cDNA probe obtained from partially purified beta-casein mRNA. The supernatant markedly stimulated beta-casein gene transcription but not 28S rRNA transcription. The same effect was obtained with other lactogenic hormones such as human growth hormone and ovine placental lactogen but was not observed with bovine growth hormone, insulin, parathyroid hormone, luteotropic hormone, or epidermal growth factor. Prolactin and human growth hormone were totally inactive when added directly to nuclei. The factor stimulating beta-casein gene transcription was also generated by membranes containing prolactin receptors such as those from liver, ovary, adrenals, and brain but not by membranes from heart, lung, and muscle, which do not bind prolactin. The factor stimulated beta-casein transcription when added to mammary nuclei from pseudopregnant or bromocryptin-treated lactating rabbits, in which the transcription rate is submaximal, but was ineffective on mammary nuclei prepared from untreated fully lactating rabbits. The factor was unable to induce beta-casein gene transcription in nuclei isolated from rabbit liver and reticulocytes. The factor did not stimulate the transcription of globin genes in nuclei isolated from reticulocytes or the transcription of mammary "housekeeping" genes evaluated by a cDNA probe prepared from total mRNA isolated from an unstimulated mammary gland. The transcription of beta-casein genes was abolished by adding alpha-amanitin to the medium in the presence or in the absence of the factor, indicating that the generation of mercurated beta-casein mRNA sequences depended upon the transcriptional activity of RNA polymerase II. The addition of the factor to the incubation mixture did not enhance total and alpha-amanitin-sensitive RNA synthesis. These data suggest that the binding of prolactin to its receptor in vitro induces the formation of a second messager, which specifically stimulates the transcription of prolactin-sensitive genes.

Animals↗

PubMed1981

[Thyroglobulin gene expression in cultured thyroid cells: regulation by thyrotropin].

The influence of thyrotropin (TSH) on thyroglobulin (Tgb) biosynthesis has been studied porcine thyroid cells maintained in primo culture. After 4 days of culture and 1-hr labelling with 35S Met, Tgb specific radioactivity was 10 times higher in TSH-treated cells than in control cells. The levels at which TSH action occurred have been described. i) In TSH cell extracts, 70 p. 100 of the Tgb mRNA sequences, quantified by hybridization to a specific 3H cDNA probe, were located in highly polymeric polysomes, while 70 p. 100 of the Tgb mRNA in the control extracts were in light polysomes. This suggests that TSH specifically or non-specifically stimulates the initiation of translation on Tgb mRNA. ii) The relative amount of Tgb mRNA in the cytoplasm of TSH-treated cells was 3 times higher than in the control cells. This correlates with the fact that Tgb biosynthesis relative to total protein synthesis increased similarly, indicating that TSH controls relative Tgb biosynthesis by specifically regulating cytoplasmic Tgb mRNA content. iii) The nuclear RNA of TSH-treated cells contained 2.2 times more Tgb mRNA sequences than the nuclear RNA of control cells, showing that TSH specifically regulates Tgb gene expression at the nuclear level (transcription or stabilization).

Animals↗

PubMed2002

cDNA sequences and mRNA levels of two hexamerin storage proteins PinSP1 and PinSP2 from the Indianmeal moth, Plodia interpunctella.

In insects, storage proteins or hexamerins accumulate apparently to serve as sources of amino acids during metamorphosis and reproduction. Two storage protein-like cDNAs obtained from a cDNA library prepared from fourth instar larvae of the Indianmeal moth (Plodia interpunctella) were cloned and sequenced. The first clone, PinSP1, contained 2431 nucleotides with a 2295 nucleotide open reading frame (ORF) encoding a protein with 765 amino acid residues. The second cDNA, PinSP2, consisted of 2336 nucleotides with a 2250-nucleotide ORF encoding a protein with 750 amino acid residues. PinSP1 and PinSP2 shared 59% nucleotide sequence identity and 44% deduced amino acid sequence identity. A 17-amino acid signal peptide and a molecular mass of 90.4 kDa were predicted for the PinSP1 protein, whereas a 15-amino acid signal peptide and a mass of 88 kDa were predicted for PinSP2. Both proteins contained conserved insect larval storage protein signature sequence patterns and were 60-70% identical to other lepidopteran larval storage proteins. Expression of mRNA for both larval storage proteins was determined using the quantitative reverse transcription polymerase chain reaction method. Only very low levels were present in the second instar, but both mRNAs dramatically increased during the third instar, peaked in the fourth instar, decreased dramatically late in the same instar and pupal stages, and were undetectable during the adult stage. Males and females exhibited similar mRNA expression levels for both storage proteins during the pupal and adult stages. The results support the hypothesis that P. interpunctella, a species that does not feed after the larval stage, accumulates these two storage proteins as reserves during larval development for subsequent use in the pupal and adult stages.

Amino Acid Sequence↗

PubMed1989

Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity.

RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.

Amino Acid Sequence↗

PubMed1983

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.

Adenovirus Infections, Human↗

PubMed1984

Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions.

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.

Albumins↗

PubMed1980

A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP.

The plasmid pDd812 contains the DNA copy of an mRNA sequence from Dictyostelium discoideum that undergoes first an increase and then a decrease in concentration during the first few hours of differentiation. We have recently shown that the mRNA sequence complementary to pDd812 encodes discoidin I, a developmentally regulated lectin that may play a role in cellular cohesion. By using pDd812 as a hybridization probe, we found that addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of high external concentrations of cAMP is known to increase the intracellular concentration to a level normally found later in development. This natural increase in cAMP concentration occurs at the time during development when transcription of the discoidin I gene ceases. We suggest, therefore, that changes in the intracellular concentration of cAMP act at the level of transcription to control gene expression during development. This hypothesis is supported by our observation that several poly(A)+RNA sequences that normally accumulate after transcription of the discoidin I gene has ceased are synthesized prematurely in cells exposed to exogenous cAMP.

Adenosine Monophosphate↗

PubMed1989

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine.

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.

Amino Acid Sequence↗

PubMed1988

Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori.

A group of structurally related proteins, referred to as '30K proteins', accumulate in a stage-dependent fashion in the larval hemolymph of the silkworm, Bombyx mori. To investigate the regulatory mechanisms of the 30K protein biosynthesis in the fat body, mRNA sequences coding for these plasma proteins were cloned and their structures and expression during the larval development were studied. Five plasmid clones, each bearing a distinct mRNA sequence coding for the 30K protein component, were isolated from a cDNA library constructed from the fat body poly(A)RNA of the mid-fifth instar larvae. These clones were remarkably similar to each other with respect to both the nucleotide and the amino-acid sequences. Each clone carried an open reading frame for amino-acid residues 251 to 264, with a typical signal sequence for transmembrane secretion at the deduced amino-terminal domain. Northern blot hybridization provided evidence that the developmental change in the amount of 30K protein mRNAs in the fat body reflects well that of the hemolymph concentration of 30K proteins, indicating that the biosynthesis of 30K proteins is developmentally regulated at a transcriptional or post-transcriptional level in the fat body.

Amino Acid Sequence↗