Erythroid expression and DNAaseI-hypersensitive sites of the carbonic anhydrase 1 gene.

The carbonic anhydrase 1 gene is expressed in adult human and mouse erythroid cells and colon epithelia from two distinct promoters. We have explored the erythroid promoter for cis-acting sequences involved in transcription using DNAaseI as a probe. Two DNAaseI-hypersensitive sites (DHS-1 and DHS-2) have been identified in the distal erythroid promoter in CA1-expressing erythroleukaemic cells. These sites are present at low levels in K562 cells, which have a foetal/embryonic phenotype and do not express CA1. DHS-1 and DHS-2 are not present in non-erythroid cells, including colon cells, which express CA1 from the proximal colon promoter. DHS-1 and DHS-2 were also generated in an heterologous CA1 gene containing 5 kb of erythroid promoter sequence after transfection into erythroid cells, including K562 cells. These transfection studies showed that both this fragment, and an abbreviated 817 bp promoter fragment which contains only DHS-1, were sufficient to confer erythroid-specific expression to a reporter gene. These promoters were active in cell lines expressing CA1 and in K562 cells. This latter observation implies that a developmental repressor factor is both present in K562 cells and binds to a cis-acting sequence that is absent from the sequence 5 kb upstream of the erythroid transcription start site.

Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8.

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.

The human carbonic anhydrase I gene has two promoters with different tissue specificities.

Primer extension and S1 nuclease analysis of human carbonic anhydrase I (HCA1) mRNA transcripts in erythroid and non-erythroid tissues show that the HCA1 gene has two promoters with different tissue specificities, separated by 36 kb of DNA. The promoter of the HCA1 gene which is active in non-erythroid tissue shows strong sequence similarity with a similar region in the mouse CA1 gene, but has two equally used transcription start sites.

Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage-specific trans-acting factors.

Using flow cytometric analysis of red cells from different stages of ontogeny with anti-CAI antibody, it was shown that the human carbonic anhydrase I (HCAI) gene product appears in a developmental stage-specific manner. Virtually no CAI protein was detectable in fetal red cells prior to birth. However, at about the time of normal delivery (40 weeks gestation) CAI production is switched on. The proportion of cells containing CAI reaches adult levels during the second half of the first year of life. Northern analysis suggests that the appearance of CAI protein results directly from the activation of the gene and the production of new mRNA. A transient heterokaryon system was set up by fusing the erythroleukaemic cell lines MEL C88 (a mouse cell line in which CAI is expressed) and K562 SAI (a human cell line with an embryonic/fetal phenotype, not expressing CAI). SP6 RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This shows the developmental stage-specific expression of HCAI to be regulated by trans-acting factors.

Structure and methylation patterns of the gene encoding human carbonic anhydrase I.

The gene (CAI) encoding human carbonic anhydrase I (CAI) has been isolated and shown to have a total length of 50 kb. Some 36 kb of this consists of a large intron separating the erythroid-specific promoter from the coding region. A small (54 bp) noncoding exon from within this intron is occasionally found in transcripts. Two different polyadenylation sites have been found, the most distal of which is the most commonly used. Methylation levels near the promoter differ widely in cell lines. In CAI-expressing cells, a region of DNA near the promoter is demethylated in a generally highly methylated background. Surprisingly, non-CAI-expressing cell lines show much lower levels of methylation.

Multiple GF-1 binding sites flank the erythroid specific transcription unit of the human carbonic anhydrase I gene.

Six potential GF-1 sites which bind an erythroid factor are present in the 5' and 3' regions flanking the erythroid-specific transcription unit of the human carbonic anhydrase I (HCAI) gene. When two of these sites are placed upstream of a minimal eukaryotic promoter they confer up-regulated expression in erythroid over non-erythroid cells. The presence of the erythroid factor in TPA-treated HEL cells in which the level of HCAI transcript has greatly decreased and in non-HCAI-expressing K562 cells suggests that in these cases the presence of the factor is not sufficient for HCAI expression.

Regional localization of carbonic anhydrase genes CA1 and CA3 on human chromosome 8.

The human carbonic anhydrase isozymes represent a family of homologous proteins which are important in respiratory function, fluid secretion, and maintenance of cellular acid-base homeostasis. Using somatic cell genetic techniques we have mapped two of the CA genes (CA1 and CA3) to human chromosome 8. In situ hybridization data demonstrates that both CA1 and CA3 map to the same region (q13-q22) of chromosome 8.

DNA sequences which influence the selection and efficient utilisation of transcriptional start sites of a eukaryotic gene by RNA polymerase II in vitro and in vivo.

Experiments are described which probe the relationship between three sequence elements which make up the eukaryotic RNA polymerase II promoter. A cloned eukaryotic gene, from which the TATA-box and 400 base pairs of 5'-flanking sequence has been deleted, is still transcriptionally active in vivo (following its transfection into cultured mammalian cells) and in vitro. Deletion has appropriately positioned a cluster of five TATA box-like sequences upstream from multiple potential cap sites. Which cap sites are actually used can be predicted from the DNA sequence of TATA box-like sequences and their spatial relationship with respect to possible transcriptional start sites, although there appears to be some difference in cap site utilisation in vitro and in vivo. Data suggest that deletion has also removed "upstream" sequences which affect promoter function.

Assignment of the gene determining human carbonic anhydrase, CAI, to chromosome 8.

A cDNA clone complementary to the mRNA encoding the rabbit erythrocyte specific carbonic anhydrase, CAI, has been used as probe for human CAI sequences in the analysis of DNA from panels of rodent/human somatic cell hybrids. The presence of the human CAI gene in all hybrids correlates with the presence of chromosome 8. Together with published mapping data, this assignment indicates that three CA loci are situated on chromosome 8.

The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo.

We have studied how small changes in the distance between the TATA-box and cap site affect transcription of a eukaryotic gene in vitro and in vivo. The trout protamine gene TPG-3 [Gregory et al. (1982) Nucl. Acids Res. 10, 7581-7592] is a good model for such a study as it has (i) a consensus TATA-box 32 base pairs (bp) upstream from an A-residue which is the natural cap site (designated +1) (ii) two further A-residues at -5 and +5, providing alternative transcriptional start sites which are in significantly different sequence environments and (iii) a unique AvaII restriction site immediately downstream from the TATA-box which is ideal for the insertion or deletion of up to 3bp. Transcripts of the wild type and mutant genes were generated in vitro using a HeLa whole cell extract or 'in vivo' by transient expression following their transfection into HeLa cells. These 'spacer' mutations did not affect the efficiency of transcription of the gene in vitro but they did affect the selection of transcriptional start site both in vitro and 'in vivo'. Analysis of 5'-ends by S1-mapping and primer extension showed that the A-residue(s) selected are those which, by insertion or deletion, come to lie on the same face of the DNA double helix as the TATA-box, although the DNA sequence in the immediate vicinity of the potential start sites influences their utilisation. Comparison of the TPG-3 wild type transcripts in these experimental systems with natural mRNA suggests that cap site selection is more stringent in the developing trout testis.

Transcription of a cloned rainbow trout protamine gene is accurately initiated following transfection into HeLa cells but the majority of the transcripts fail to polyadenylate at the correct site.

The expression of a cloned trout protamine gene transfected into mammalian cells in culture has been studied. This small intronless gene has a consensus TATA-box, a classical AATAAA sequence and the cap and polyadenylation sites are separated by only 228 base pairs (Gregory et al., ref 10). When 1kb of cloned trout genomic DNA containing this sequence was introduced into HeLa cells, S1-mapping showed that transcripts of the protamine gene were accurately initiated at the in vivo cap site but were not polyadenylated at the authentic 3'-site. Replacement of the 3'-end of the protamine transcription unit with a fragment of SV40 containing the small-t intron and early polyadenylation site resulted in only a modest increase in transcript levels over the wild-type gene in HeLa cells. However, transcripts of a fusion gene in which the 5'-end of the protamine gene was replaced by the SV40 early promoter were present at extremely low levels in transfected COS cells. The data are discussed in the context of the involvement of RNA processing events in the stabilisation of eukaryotic gene transcripts.

Eukaryotic ternary transcription complexes: transcription complexes of RNA polymerase II are associated with histone-containing, nucleosome-like particles in vivo.

Using a psoralen crosslinking, radioactive labelling technique, we have previously been able to study ternary transcription complexes containing DNA-dependent RNA polymerases I and II which are released from rat liver nuclei by endogenous nuclease digestion [Sargan and Butterworth, refs 1 and 2]. Although the DNA component of these complexes was found to have a 'nucleosome-like' size profile and although the experimental conditions for autodigestion were designed to minimise histone rearrangement, it is necessary to provide further evidence that the periodicity of nuclease cutting around these transcription complexes is conferred by histones. Studies using secondary nuclease digestion of the released transcription complexes now show a digestion barrier characteristic of that conferred by nucleosomal histones which is lost if histones are removed from the complexes. Furthermore, antibodies raised against histones are effective in precipitating transcription complexes of RNA polymerase II and, to a lesser extent, of RNA polymerase I. The data suggest that, in rat hepatic tissue, transcription complexes are in very close proximity (within a few hundred base pairs) of histone-containing, nucleosome-like particles in vivo.

Cloned cDNA for rabbit erythrocyte carbonic anhydrase I: A novel erythrocyte-specific probe to study development in erythroid tissues.

Present understanding of gene expression in erythropoietic tissues is derived solely from studies of the globin genes. Of the three distinct carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) isozymes, carbonic anhydrase I is erythrocyte-specific and, in humans, is under developmental control. The appearance of carbonic anhydrase I in the erythrocyte late in fetal life follows closely the gamma- to beta-globin switch. In order to study the expression of this erythrocyte-specific nonglobin protein, we set out to isolate a cloned carbonic anhydrase I cDNA. A mixture of 17-base-long synthetic oligonucleotides was used as an in situ hybridization probe to screen a rabbit reticulocyte cDNA library. Two clones were isolated, and the complete nucleotide sequence of the clone with the largest insert was determined and shown to code for carbonic anhydrase I. This clone, designated pRCAI, is near full length and has provided the 40% of the amino acid sequence of rabbit carbonic anhydrase I, which was not known hitherto. The deduced primary structure has revealed potentially significant changes in the vicinity of the active site of the rabbit carbonic anhydrase I when compared with carbonic anhydrase I and II sequences from other species.

The generation of a single nick per plasmid molecule using restriction endonucleases with multiple recognition sites.

Some restriction endonucleases generate a single-stranded nick at their recognition sequences in the presence of ethidium bromide (EtBr). This nick can then be extended to a single-stranded gap in which mutations can be introduced by a variety of techniques. To date, the templates used in these studies have largely contained a single recognition site for a given enzyme. Therefore, we have extended these studies to twelve enzymes for which multiple recognition sites exist in the template and show that, under appropriate conditions, one single-stranded nick is introduced per plasmid molecule.

A comparison of the promoter strengths of two eukaryotic genes in vitro reveals a region of DNA that can influence the rate of transcription in cis over long distances.

We have compared the strength of a trout protamine gene promoter with that of the mouse beta major-globin gene by analysing the relative levels of run-off transcripts produced in a single mammalian in vitro transcription reaction. When the promoters are introduced on separate recombinant plasmids, the protamine transcripts are synthesised with much greater efficiency than those originating from the globin cap site. This enhanced transcription of the protamine gene is again observed when the promoters are applied as separate DNA fragments derived from the same recombinant plasmid. However, when the promoters are linked on a DNA fragment that includes 7 kb of DNA separating the initiation sites, then there is a marked reduction in the protamine signal relative to the globin. Deletion of a region of this fragment that contains the sequences flanking the globin gene at positions -335 to -1400 restores the enhanced protamine gene expression to the levels observed when the promoters are carried on separate DNA fragments.

The localisation of the 5'-termini of in vivo transcripts of a cloned rainbow trout protamine gene.

The mRNA start site of a cloned rainbow trout protamine gene (TPG-3) has been localised using S1-nuclease mapping and primer extension of in vivo synthesised trout testis poly A+-RNA. The presumptive cap site occurs within an AT-rich region, only 14 nucleotides from the start of the protein-coding sequence. Transcription of this protamine gene in vitro, using the Hela whole-cell extract system, generates products initiated at the same nucleotide as that used in vivo. In vitro transcription is abolished by deletion of sequences between -20 and -48, within which is a canonical TATA-box having an llbp homology with the strong chick conalbumin and Adenovirus-2 major late promoters (CTATAAAAGGG).

A possible novel interaction between the 3'-end of 18 S ribosomal RNA and the 5'-leader sequence of many eukaryotic messenger RNAs.

A novel interaction between the 5'-untranslated region of eukaryotic messenger RNAs and non-contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3'-terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5'-leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A-U-C-C-A-C-C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG-codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.

Eukaryotic ternary transcription complexes. II. An approach to the determination of chromatin conformation at the site of transcription.

Digestion of rat liver nuclei by endogenous nucleases or micrococcal nuclease releases a chromatin fraction containing RNA polymerases I and II bound to DNA fragments in ternary transcription complexes. To label the DNA in these transcription complexes, the polymerases were allowed to add radioactively labelled ribonucleotides in vitro to in vivo-initiated RNA chains. During this transcription step, nucleic acids were photochemically cross-linked using 8-methoxypsoralen. Nucleic acids in transcription complexes were then sized by gel electrophoresis. Under conditions where RNA polymerases I and II were active in vitro, most of the labelled DNA was found in a series of fragments of sizes which were multiples of approximately 200 base-pairs. When polymerase I alone was active, the smallest member of this series carried the bulk of the label; when polymerase II also was active, a significant proportion of the label was carried on the dimer and higher oligomers. Proteins other than polymerase alone are shown to be responsible for the pattern of DNA fragments protected from nucleases. Therefore active RNA polymerases I and II in vivo are in close proximity to structures protecting DNA fragments, the sizes of which are similar to those found in nucleosomes. We have yet to establish that these structures are composed of histones.

Eukaryotic ternary transcription complexes. I. The release of ternary transcription complexes of RNA polymerases I and II by the endogenous nucleases of rat liver nuclei.

Autodigestion of rat liver nuclei in magnesium-containing buffers leads to the release of about 80% of DNA-dependent RNA polymerases I and II, together with 4 to 8% of the DNA. The RNA polymerases are at least partially DNA bound as judged by the effect on in vitro transcription of (1) Actinomycin D, (2) preirradiation of the enzymes in the presence of 8-methoxypsoralen and (3) heparin. The released DNA migrates as ladders of nicked nucleosome-size fragments on electrophoresis. On sucrose gradients, most RNA polymerase activity sediments as two peaks: one slightly smaller than the 11S mononucleosome and the other with the dinucleosome. The released material can act as a source of ternary transcription complexes for further structural studies.