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Biomedical subjects

I Boime

Publications and source records attributed to I Boime.

At least 145 records · Page 8Linked to original sources
PubMed1977

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term.

DNA↗
PubMed1976

Synthesis of human placental lactogen messenger RNA as a function of gestation.

It was shown previously that 4 to 5 times more human placental lactogen (hPL) was synthesized in cell-free extracts from term placentae than in comparable extracts prepared from first trimester tissue. In an attempt to define what accounts for this differential rate of synthesis RNA was prepared from first trimester and term placentae. Following purification through an oligo(dT)-cellulose column, these RNA preparations were tested for their ability to direct the synthesis of the hPL precursor in the wheat germ cell-free system. With similar amounts of first trimester and term mRNA, the overall efficiency as defined by the stimulation of total amino acid incorporation was comparable. However, there was approximately 4 times more hPL synthesized in the presence of term RNA. This was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic fingerprinting. The peak of the hPL precursor messenger activity sedimented at 12 to 13 S on sucrose gradient. Analysis of the RNA by formamide-polyacrylamide gel electrophoresis further supported this value. The data indicate that the increased synthesis of hPL at term reflects greater levels of hPL mRNA in term tissue than in first trimester tissue. The data also show that the overall in vivo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in the greater proportion of hPL synthesized per g of tissue. The latter results from the continual differentiation of the placenta occurring throughout gestation.

Female↗
PubMed1976

Isolation of a biologically active messenger RNA: preparation from fish pancreatic islets by oligo(2'-deoxythymidylic acid) affinity chromatography.

RNA was extracted from the pancreatic islets of channel catfish in the presence of the ribonuclease inhibitor, diethyl pyrocarbonate (oxydiformate). High molecular weight RNA was observed on sucrose gradient analysis. enrichment of mRNA was achieved by oligo(2'-deoxythymidylic acid)-cellulose affinity chromatography. The mRNA fraction stimulated incorporation of [35S]methionine into protein up to 30-times the background in the wheat-germ cell-free system. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed two major proteins corresponding to molecular weights of 27 000 and 11 000. These proteins were not observed in the absence of mRNA or in the presence of mRNAs from other tissues. They were also synthesized in the ascites tumour cell-free system. No protein co-migrating with proinsulin or insulin was detected in either the ascites or wheat-germ cell-free systems. Pancreatic islet slices also synthesized the proteins of 27 000 and 11 000 molecular weight and smaller ones as well.

Animals↗
PubMed1976

mRNA-dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormone form in ascites cell-free extracts.

Messenger RNA derived from term placenta directs the synthesis of human placental lactogen (hPL, molecular weight 22,200) in an ascites 30,000 X g post-mitochondrial supernate (S-30). When the S-30 is fractionated into ribosome and cell-sap (S-100) fractions, and these are recombined for incubation, term placental mRNA directs the synthesis of a protein with a molecular weight of 25,000. This protein contains authentic hPL tryptic peptides. This suggested that during the separation of ribosomes and S-100 a component responsible for cleavage was lost. A 1.0 M sucrose cushion was used for the preparation of ribosomes and S-100 and membranous material accumulated at the sucrose interphase. When this membrane fraction was added back to the ribosome-S-100 system only hPl was formed. Cleavage was greatest when membranes were added within the first few minutes of incubation. In a run-off system composed of term polysomes, ascites S-100, and the inhibitor of initiation, pactamycin, the 25,000 molecular weight material, referred to as pre-hPL, was also synthesized. These data strongly suggest that (i) pre-hPL is an authentic percursor to hPL, (ii) cleavage of the precursor primarily occurs on nascent, ribosome-bound peptide chains, and (iii) pre-hPL is the primary gene product.

Cell-Free System↗
PubMed1976

The cell-free synthesis of the alpha subunit of human chorionic gonadotropin.

The synthesis of the alpha subunit of human chorionic gonadotropin (hCG) was demonstrated in a cell-free system composed of polysomes derived from first trimester placenta and cell sap prepared from ascites tumor cells. The in vitro synthesized proteins labeled with [35S]methionine were shown to have at least 4 tryptic peptides that co-migrated with the same peptides from authentic hCG. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed the synthesis of a discrete protein, migrating with an apparent molecular weight of about 17,000, which contained methionine-labeled tryptic peptides found in the alpha subunit. The level of radioactivity in these tryptic peptides was five times greater with polysomes from first trimester placentae than with those from term placentae. The efficiency of total protein synthesis in both cases was about the same. These data strongly suggest that the decrease in blood levels of hCG after the first trimester is caused by a selective decrease in the rate of synthesis of the hormone.

Amino Acids↗
PubMed1974

The synthesis of human placental lactogen by ribosomes derived from human placenta.

In a very active cell-free system containing polysomes derived from human placenta and a cell-sap fraction prepared from ascites tumor cells, the synthesis of the hormone human placental lactogen (HPL) was detected. The identification was based on the following: (a) The in vitro synthesized protein labeled with [(35)S]methionine migrated at the same rate as authentic HPL on sodium dodecyl sulfate-polyacrylamide gels and (b) tryptic fingerprint analysis of the labeled protein yielded peptides having the same mobilities as seen with the same analysis of purified HPL. The amount of HPL synthesized in a cell-free system containing polysomes derived from term placenta was about 10% of the total proteins synthesized and in a comparable system containing first trimester ribosomes the level of synthesis was about 5%. These data suggest the potential for quantitating the HPL mRNA activity as a function of the period of gestation and for isolating the mRNA itself.

Animals↗
PubMed1972

Translation of bacteriophage Q messenger RNA in a murine Krebs 2 ascites tumor cell-free system.

Qbeta is a small bacterial virus whose three genes are encoded in a single-stranded molecule of RNA. This RNA serves directly as the Qbeta message. Here we describe conditions under which RNA corresponding to the coat cistron of this bacterial virus is translated in a system derived from mammalian cells. Translation of the bacterial virus messenger RNA is less effective than that of mammalian globin messenger RNA, but is somewhat enhanced by mild alkali treatment of the messenger. The synthesized product when subjected to electrophoresis migrates with authentic Qbeta coat protein and yields tryptic peptides that correspond to those derived from the Qbeta coat protein.

Amino Acids↗
PubMed1971

Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system.

Small amounts of encephalomyocarditis virus RNA direct a 50-fold increase in amino acid incorporation, in appropriately supplemented ascites tumor cell extracts, under conditions that give rise to authentic viral polypeptides. Incorporation in these crude extracts has a novel characteristic, namely, that it is almost entirely dependent upon the addition of exogenous tRNA. Further, this incorporation is restricted in that tRNA derived from ascites tumor cells or from rat liver permits translation of viral RNA, whereas tRNA from yeast or Escherichia coli does not. These translational barriers are due, at least in part, to an incompatibility between the tRNA of yeast and E. coli and the aminoacyl-tRNA synthetases of the ascites tumor cell. A more extensive basis for this incompatibility is suggested, however, by the failure of the E. coli aminoacyl-tRNA synthetases to restore viral RNA-directed protein synthesis in the presence of tRNA from E. coli, although the coli synthetases fully restore the poly(U)-directed synthesis of polyphenyl-alanine. The possible role that unique or favored codon classes might play in this restriction is considered, together with the implications of the observed requirement for tRNA.

Alanine↗