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Biomedical subjects

A Colman

Publications and source records attributed to A Colman.

At least 73 records · Page 4Linked to original sources
PubMed1985

The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes.

Synthetic RNAs coding for chicken lysozyme, calf preprochymosin and Xenopus globin were transcribed in vitro using Sp6 RNA polymerase. The effects of capping and adding a poly(A) tail on the stability, movement and translation of these RNAs in Xenopus oocytes was examined. Capping and polyadenylation increased stability of the transcripts, with at least 40% remaining intact 48 h after injection into oocytes. Capped poly(A)- transcripts moved more rapidly in oocytes than either capped poly(A)+ transcripts or naturally occurring mRNAs. The translational efficiency of most of the synthetic RNAs in oocytes increased with both capping and polyadenylation. The exception was one Xenopus globin transcript which had an unusual 3' end of 20As and 30Cs, where further polyadenylation decreased translational efficiency. Polyadenylation was essential for detectable expression of the synthetic RNAs in cultured cells, but decreased translation of the synthetic RNAs in vitro.

Animals↗
PubMed1985

Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes.

Influenza virus nucleoprotein (NP), synthesized in Xenopus oocytes after injection of cloned NP cDNA, enters and accumulates in the nucleus. We have used in vitro mutagenesis of this cDNA to study the cellular distribution of mutated NP polypeptides. Mutants lacking amino acids 327-345 of wild-type NP enter the nucleus but do not accumulate there to the same extent as the wild-type protein, suggesting that this region has a role in nuclear accumulation. This possibility is further strengthened by similar studies involving the production of fusion proteins in which various amino-terminal sequences of the NP gene are fused to the complete chimpanzee alpha 1-globin sequence: when globin cDNA was injected into and expressed in oocytes the protein remains exclusively in the cytosol; however, when the globin cDNA is fused to a portion of NP cDNA that includes the region encoding amino acids 327-345, the resulting fusion protein enters and accumulates in the nucleus. Fusion proteins lacking this region of the NP enter but do not accumulate in the nucleus.

Animals↗
PubMed1985

Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes.

The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.

Animals↗
PubMed1985

Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati.

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.

Animals↗
PubMed1985

Location of influenza virus M, NP and NS1 proteins in microinjected cells.

When microinjected as cloned DNA, the nucleoprotein (NP) of influenza virus A/NT/60/68 (H3N2) accumulated in the nuclei of Xenopus laevis oocytes, and cultured cells of rodent and primate origin. This accumulation appeared to be specific and a property of the NP itself (or conceivably NP in association with unknown cellular constituents) since no other influenza virus components were present in DNA-injected cells. In the oocyte nucleus, clonally derived NP achieved an eightfold concentration over that in the cytoplasm. Such NP was full-length as judged by its mobility during PAGE and had the native conformation of H3N2 virus NP according to its reaction with a panel of monoclonal antibodies. NP appeared to be in the soluble fraction of the nucleus as it did not sediment under conditions which removed particulate matter from nuclear extracts. Microinjection of extracts of chick embryo fibroblast cells infected with A/FPV/Rostock/34 (H7N1) showed that exogenous NP had an affinity for the nucleus similar to that synthesized intracellularly from cloned NP DNA. This conclusion was supported by an experiment in which cloned NP from the oocyte nucleus re-entered the nucleus after injection into the cytoplasm of fresh oocytes. Injection of mRNA, extracted from chick embryo fibroblast cells infected with A/FPV/Rostock/34, into oocytes directed the synthesis of the viral proteins M (Mr 28 000), and NS1 (Mr 27 000) as well as NP (Mr 56 000). While NP from this source concentrated in the nucleus as before, M merely associated with the nucleus without exceeding the cytoplasmic level. Even more remarkable was NS1; although in injected cells this protein is concentrated in nucleoli, in microinjected oocytes its nuclear concentration was threefold less than that in the cytoplasm, despite the very large number (greater than 1500) of nucleoli present in Xenopus oocytes. It seems likely that the karyophilic nature of M and NS1, unlike that of NP, is a property not of the proteins themselves, but of a complex which they form with some other product of the infected cell. These findings were repeated when extracts from infected chick embryo cells containing NP, M and NS1 proteins radiolabelled in vivo, were injected into the cytoplasm of oocytes.

Animals↗
PubMed1984

Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes.

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.

Amino Acid Sequence↗
PubMed1984

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes. Identification of an ovalbumin signal sequence.

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee alpha-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin "signal" sequence is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.

Amino Acid Sequence↗
PubMed1984

Xenopus oocytes can synthesise but do not secrete the Z variant of human alpha 1-antitrypsin.

Human liver mRNA was prepared from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and alpha 1-antitrypsin identified by immunoprecipitation. The normal M variant of alpha 1-antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M alpha 1-antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.

Animals↗
PubMed1984

Medication usage, emotional disturbance, and pain behavior in chronic low back pain patients.

Examined chronic low back pain outpatients (N = 126) at a university hospital outpatient back clinic. Prior to their orthopedic examination, patients were given the MMPI, the Pain Experience Questionnaire (a modified form of the McGill Pain Questionnaire), and a medical history form that indicated onset of injury, number of surgeries, functional limitations, and use of medications. Four patterns of drug usage emerged with sufficient number to permit statistical analysis: (1) none; (2) aspirin-type medications; (3) single narcotics alone; and (4) narcotics combined with other medications. Group 4 patients scored significantly higher on the Hypochondriasis and Hysteria scales than the others, whereas Group 3 patients scored higher on the PD (acting out) scale. Group 4 patients reported more intense pain and chose more affective terms (punishing/terrorizing/sickening) in describing their pain than the other three groups. Group 1 patients reported significantly less limitation in walking, running, and climbing.

Adult↗
PubMed1983

Post-translational fate of variant MOPC 315 lambda chains in Xenopus oocytes and mouse myeloma cells.

The post-translational fates of three immunoglobulin lambda chain variants of MOPC 315 were investigated in mouse plasmacytoma cell lines and in mRNA-microinjected Xenopus oocytes. Quite unexpectedly we found that one non-secretory variant chain (lambda-43) underwent extensive post-translational N-glycosylation: however the presence of the oligosaccharide moiety did not account for the nonsecretory phenotype nor did it affect the rate of degradation of this lambda chain. Another variant chain (lambda-47) at first believed to be non-secretory, was found to be secreted from oocytes at a very low level, but mostly as a lambda-lambda dimer. In myeloma cells a low level of lambda-47 chain was secreted and again lambda-lambda dimers were the favoured secretory form. The secretory lambda-48 chain also formed lambda-lambda dimers, whereas lambda-43, which was never secreted, was only found as a monomeric lambda chain in both oocytes and myeloma cells. A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (gamma 1) chain mRNA and MOPC 315 lambda chain mRNAs. The wild type lambda chain (lambda-48) was able to assemble with the gamma chain in a covalently bound tetramer (gamma gamma lambda lambda). The variant lambda-47 chain was also able to form gamma gamma lambda lambda tetramers, whereas the lambda-43 was not, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted. These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell-type specific.

Animals↗
PubMed1983

Analysis of histone H5 and globin mRNAs in chick-embryo development.

Transcripts from histone H5 and alpha and beta chicken globin genes were analysed in total RNA extracted from 2-6-day-old chick embryos. Denatured RNA, separated on gels, was transferred to either aminophenylthioether (APT) paper or to nitrocellulose and was detected with 32P-labelled recombinant DNA probes. Good resolution was achieved with methyl mercury hydroxide gels and aminophenylthioether transfer, allowing estimation of H5 and alpha and beta globin mRNA sizes. With glyoxal gels and nitrocellulose transfer, alpha and beta globin mRNAs were not resolved, but an increased sensitivity of detection of at least 10-fold was achieved. Levels of H5 and alpha globin mRNAs increased in parallel over 2-6 days. The appearance of definitive red cells in 6-day embryos was indicated by detection of adult beta globin transcripts at this stage.

Animals↗
PubMed1983

The oocyte as a secretory cell.

Xenopus laevis oocytes secrete a large variety of foreign secretory proteins after the microinjection of mRNA or DNA. Two classes of such proteins are discussed in detail. These are the chick oviduct proteins ovalbumin and lysozyme, and the mouse MOPC 21 immunoglobulin. The injection of mRNAs for mouse immunoglobulin heavy or light chain leads to the synthesis, segregation, but not secretion of the encoded proteins unless the two mRNAs are simultaneously or sequentially injected into the same oocytes. Chicken ovalbumin and lysozyme are synthesized and secreted from oocyte after the injection of either oviduct mRNA or cloned DNA (ovalbumin). The secreted lysozyme is exported considerably faster than ovalbumin; however, 40% of the lysozyme synthesized cannot be secreted and, after fractionation of oocytes on sucrose gradients, is found in a higher density position than ovalbumin. No competition at the level of secretion or translation was noted when different amounts of immunoglobulin and ovalbumin mRNAs were injected into oocytes. However, the co-injection of ovalbumin mRNA and mRNAs encoding anti-ovalbumin immunoglobins resulted in the formation of a complex of the two types of protein within the oocyte. In these circumstances, secretion of the immunoglobulin was severely reduced.

Animals↗