Gene transfer for therapy and prophylaxis of HIV-1 infection.
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M L Rohrbaugh
Publications and source records attributed to M L Rohrbaugh.
Transcription unit of the rabbit beta 1 globin gene.
We have hybridized pulse-labeled nuclear transcripts to cloned DNA fragments from the rabbit beta-like globin genes to determine the developmental timing, extent, and asymmetry of their transcription. The fetal-adult gene beta 1 was transcribed in fetal liver but not embryonic nuclei, whereas genes beta 3 and beta 4, which encode embryonic globin polypeptides, were transcribed only in embryonic nuclei. This shows that the switch from embryonic to fetal-adult globin production in rabbits is accomplished primarily by differential transcription of the beta-like globin genes. Gene beta 1 was subdivided into M13 subclones and tested for hybridization to nascent RNA. The nucleotide sequence of the 3' flanking region of gene beta 1 was also determined for 2,447 base pairs past the polyadenylation [poly(A)] site. No transcripts were found 5' to the cap site, but asymmetric transcription of gene beta 1 proceeded at a high level through the gene and past the poly(A) addition site for 603 nucleotides. The level of transcription declined after this, gradually dropping through the next 568 nucleotides. No polymerases were found on a fragment that begins 1,707 nucleotides past the poly(A) site; this fragment was part of a segment of repetitive DNA. These data show that the transcription unit of gene beta 1 begins at or near the cap nucleotide and extends at least 1,171 but no more than 1,706 nucleotides past the poly(A) addition site. The DNA segment that precedes the region of declining transcription contained an inverted repeat and encoded a short RNA transcribed by RNA polymerase II from the strand opposite the beta 1 transcript. These two features may function to attenuate the transcription of gene beta 1. An inverted repeat and a potential polymerase II transcription unit were also found in the homologous segment 3' to the human beta-globin gene. A short DNA segment close to the 3' end of the beta 1 transcription unit was transcribed more actively than the surrounding DNA, and it contained sequences that match the consensus internal control region for RNA polymerase III. This DNA segment may contain a separate polymerase III transcription unit. A member of the D repeat family located 3' to gene beta 1 was not transcribed in its entirety coordinately with beta 1.
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Analysis of rabbit beta-like globin gene transcripts during development.
We have analyzed the differential expression of a family of beta-like globin genes during the development of rabbits, from four days post implantation to one week before birth. The family is composed of four genes, arranged 5'-beta 4-beta 3-psi beta 2-beta 1-3' on the chromosome; psi beta 2 is an inactive pseudogene. Using the technique of hybrid-arrested translation in vitro, we have identified the embryo-specific globin polypeptides encoded by genes beta 3 and beta 4. The beta 3 and beta 4 globins are replaced by the adult beta 1 globin halfway through gestation; this corresponds temporally with the switch in site of erythropoiesis from the embryonic yolk sac to the fetal liver. The decline in production of beta 3 globin polypeptide precedes the decline in beta 4 globin. Transcripts from genes beta 1, beta 3 and beta 4 were analyzed at progressive stages of gestation by a blot-hybridization assay and by an S1 nuclease protection assay. Mature messenger RNA and presumptive precursor RNAs from genes beta 3 and beta 4 are synthesized abundantly in embryonic erythroid cells but only at very low levels later in fetal development. Conversely, precursor and mature mRNA from gene beta 1 are found at very low levels in embryos but are abundant in fetal and adult erythroid cells. The co-ordinate appearance of precursor RNA, mRNA and polypeptide from all three active genes indicates that the primary developmental regulation of this gene family is exerted at the level of transcription. RNA species larger than the expected precursors were observed when the RNA was denatured with formaldehyde but not when methylmercury was the denaturant. These large RNAs are a formaldehyde-generated artifact, possibly a result of cross-linking globin transcripts to ribosomal RNA. We observe no extensive stable transcripts from the 5' or 3' flanking regions of these genes.
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