Human hemoglobin expression in Escherichia coli: importance of optimal codon usage.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.     

Regulation by HIV Rev depends upon recognition of splice sites

The ability of the Rev protein of HIV to regulate the cytoplasmic level of unspliced RNA from a beta-globin gene containing the Rev response element was dependent on the integrity of the 5' and 3' splice sites. A beta-globin pre-mRNA containing the Rev response element is not under regulation by Rev but is made Rev responsive by a mutation at either the 5' or 3' splice site. These mutant RNAs accumulated in the nucleus as unspliced precursors owing to recognition by splicing components. Only in the presence of Rev did these unspliced RNAs appear in the cytoplasm. Thus, regulation by Rev probably involves the dissociation of splicing components and pre-mRNA.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.     

Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells

We have examined how splicing affects the expression of a range of human and nonhuman genes in vertebrate cells. Although our data demonstrate that splicing invariably enhances the level of gene expression, this positive effect is generally moderate. However, in the case of the human beta-globin gene, splicing is essential for significant protein expression. In the absence of introns, 3' end processing is inefficient, and this appears to be causally linked to a significant decrease in the level of both nuclear and cytoplasmic 3' end-processed RNA. In contrast, splicing appears to only modestly enhance nuclear mRNA export. Consistent with this observation, intronless beta-globin gene expression was only partially rescued by the insertion of retroviral nuclear mRNA export elements. Surprisingly, in the case of the highly intron dependent beta-globin gene, the mRNA that did reach the cytoplasm was also only inefficiently translated if it derived from an intronless expression plasmid. Together, these data argue that splicing can increase gene expression by enhancing mRNA 3' end processing, and hence, mRNA production. Moreover, in the case of the highly intron-dependent beta-globin gene, splicing also significantly enhanced the translational utilization of cytoplasmic beta-globin mRNAs.