Translational regulation of the differential synthesis of nonmuscle tropomyosin isoforms.

Regulation of the differential synthesis of tropomyosin (TM) isoforms in chicken embryo fibroblasts was studied. Cell-free translation experiments with total RNA and Northern blot analyses of TM mRNAs from fractionated polysomes were performed. Unlike the major isoforms, mRNAs encoding at least one of the minor isoforms were only partially associated with the heavy polysomes. Thus, these experiments revealed that translational regulation plays a role in controlling the relative amounts of major and minor isoforms in the cell.

A high-efficiency vector for expression of foreign genes in myeloma cells.

We have constructed a high-efficiency vector for expression of genes of interest in myeloma cells. This vector is comprised of regulatory sequences from immunoglobulin (Ig) genes, including the heavy-chain enhancer, a kappa light-chain promoter and splice site, and the polyadenylation signal downstream from the kappa constant region. The expression capacity of this vector was assayed in J558L myeloma cells using human tissue plasminogen activator as a reporter gene. Stable transfectants were analyzed for protein, RNA, DNA copy number and transcription rate. Expression was compared to that of intact, transfected Ig genes and to endogenous Ig. Tissue plasminogen activator cloned into this vector was found to be expressed as efficiently as intact, transfected Ig genes, producing 1-2% of total cellular mRNA from a single copy of the gene. RNA levels and transcription rates relative to those of endogenous Ig genes were found to be about 25% and 38%, respectively. Because of its high efficiency, potential for gene amplification, and various scale-up advantages of myeloma cells, this vector-host system may yield levels of desired proteins than currently available systems.

Timing and rates of synthesis of early histone mRNA in the embryo of Strongylocentrotus purpuratus.

The level of early histone mRNA in Strongylocentrotus purpuratus changes abruptly at 6 hr of development, increasing an average of 10-fold by 9-10.5 hr and then decreasing over 2-fold by 13.5-15 hr. These changes occur when the late embryonic mRNA is still a very minor component of histone gene transcription. The exact values of increase and decrease of mRNA level vary from experiment to experiment and may reflect the conditions of embryos at different times. The instantaneous rate of synthesis of histone RNA per embryo increases from at least 47 fg/min at 6 hr to 114 fg/min at 9 hr and then drops to 29 fg/min at 12 hr. The rate of mRNA accumulation is lower: 20, 43, and 12 fg/min, respectively. On a per cell basis, however, the rate of synthesis and accumulation is highest at 6 hr and continuously decreases to 1/20 the level per cell at 12 hr. The transcriptional rates and relative mRNA increases taken together predict an average increase from 0.16 to 0.24 pg/embryo (6-10 X 10(5) molecules) per mRNA species in the egg to 1.6 to 2.4 pg/embryo (6-10 X 10(6) molecules) at 10.5 hr. The transcription rates indicate that at the maximal values we obtained, about two to three molecules of each histone RNA are made per gene copy per minute. The half-life of the histone mRNAs in the period from 6 to 13.5 hr probably varies, with the maximal turnover at about the time histone RNA level peaks. A half-life of 1.5 hr at 12 hr of development is estimated. Change in transcriptional rate per nucleus, increase in cell number, and probably a change in mRNA stability as well are therefore involved in the control of histone mRNA levels in the early embryo.

Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.