Bioassay for components regulating eukaryotic gene expression: a chromosomal factor involved in the generation of histone mRNA 3' termini.

Self Cite

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5-to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.

Section: Resultsmentioning

confidence: 96%

mentioning

confidence: 99%

Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.

Mous¹,

Stunnenberg²,

Georgiev³

et al. 1985

Self Cite

“…This approach has been used successfully to identify DNA sequences required for efficient transcription of the herpes simplex virus thymidine kinase gene (25). However, except for the elegant studies of 5S gene transcription (7,8) and a study that describes a chromosomal factor involved in transcription termination of a sea urchin histone gene (41), oocytes …”

Section: -O-acetyltransferase (Cat)mentioning

confidence: 99%

Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes.

Jones¹,

Richter²,

Weeks³

et al. 1983

The regulation of adenovirus type 5 gene expression by the Ela gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the Ela gene were coinjected into oocyte nuclei. The presence of the Ela gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the Ela gene requires the removal of intron sequences by RNA splicing. The Ela gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary Ela transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the Ela mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the Ela protein may supplant the Ela plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

“…Coinjection of DNA and cell extracts into frog oocytes has previously been used in the identification of regulatory factors for sea urchin histone genes (5,12,20) and in transcriptional studies with the adenovirus ElA protein (7,15).…”

mentioning

confidence: 99%

“…Chromatin salt-wash fractions (CSWFs) were isolated from a chicken erythroid cell line by the methods of Stunnenberg and Birnstiel (20). The cells used (ts34 AEV LSCC HD3 [2]) (AEV cells) are transformed by a temperature-sensitive avian erythroblastosis virus, and they express H5 mRNA and protein (19).…”

mentioning

confidence: 99%

H5 gene specific trans-activation by nuclear extracts from avian erythroid cells.

Wigley

Wells

1987

Nuclear extracts from chicken erythroid cells selectively stimulate transcription of the chicken histone H5 gene (and not of other chicken histone genes) after coinjection into frog oocytes. This effect is shown to involve an enhancerlike activity, and a region of the H5 gene sufficient to mediate trans-activation is defined.H5 is a linker histone variant (1, 13) which, in the chicken, is found only in erythroid cells (19). Levels of H5 protein increase during the differentiation and maturation of these cells (11,17,21), and this increase is correlated with chromatin condensation and repression of replication and transcription (17). We have previously isolated and characterized the single-copy chicken H5 gene (9) and demonstrated that transcription of this gene is accurately initiated in Xenopus oocytes (22). Our current interest lies in the transcriptional regulation of this gene.We report here our use of the frog oocyte to identify factors in erythroid-cell extracts that are involved in transcription of the H5 gene. Coinjection of DNA and cell extracts into frog oocytes has previously been used in the identification of regulatory factors for sea urchin histone genes (5,12,20) and in transcriptional studies with the adenovirus ElA protein (7,15).Effect of erythroid-cell extracts on the level of H5 transcripts in coinjected oocytes. Chromatin salt-wash fractions (CSWFs) were isolated from a chicken erythroid cell line by the methods of Stunnenberg and Birnstiel (20). The cells used (ts34 AEV LSCC HD3 [2]) (AEV cells) are transformed by a temperature-sensitive avian erythroblastosis virus, and they express H5 mRNA and protein (19).To test the fractions for possible effects on H5 gene expression, CSWF samples were coinjected, with the H5 gene and control genes, into batches of frog oocytes. The injected DNA was a mixture of pH.5/2.6 DNA, containing the H5 gene from -1,200 to +1,360 in pBR322 (Fig. la), and pH1/H23 DNA, containing two chicken histone genes, Hi and H2B (from pCH7.OE [3]), in pAT153. As shown in Fig. 2a, the CSWFs were injected into the oocyte cytoplasm, followed by nuclear injection of the DNA mixture. Oocyte manipulations were carried out according to Gurdon (6). DNA (5 to 10 ng) and CWSF (protein) (25 to 100 ng) were injected into each oocyte, and after incubation, total RNA was isolated from pooled batches of 25 oocytes (14). Figure 2b shows the result of quantitative primer extension analysis (10) of the oocyte RNA with synthetic 26-base primers. Extension on all three histone transcripts was carried out in the same reaction; each transcript generated two or more major extension products (due to cap site heterogeneity). Track 1 shows the result obtained when buffer alone was coinjected with the DNA, and track 2 shows coinjection of an AEV cell CSWF with the DNA.By comparing the two tracks, it can be seen that injection of the CSWF did not affect the level of Hi and H2B * Corresponding author.transcripts produced. In contrast, injection of the CSWF resulted in a dramatic increase (of at least 10...