Biomedical subjects
Publications and source records attributed to R Breathnach.
We present the sequence of regions of the chicken ovalbumin gene believed to be important in the control of initiation of transcription, splicing, and transcription termination or polyadenylation. Comparison with corresponding areas of other genes reveals some homologous regions which might play a role in these processes.
Mouse thymidine-kinase-negative L cells were transformed with a cloned chicken ovalbumin gene linked to the cloned thymidine kinase gene from herpes simplex virus type 1. Most thymidine-kinase-positive clones contained one or more copies of the ovalbumin gene, which were stably maintained for at least 6 months during continuous culture under selective conditions. Transcription of the ovalbumin gene was detected at a low level, producing RNA molecules that were correctly spliced and polyadenylylated by comparison with genuine ovalbumin mRNA. However, the 5' end of these RNA molecules does not correspond to that of ovalbumin mRNA.
We have determined the sequence of ovalbumin RNA (ov-mRNA) using a double-stranded cDNA (dscDNA) plasmid. We have also determined the sequence of the previously characterized exonic regions of the chicken ovalbumin gene. The comparison of these various sequences has shown that there are no additional interruptions in the mRNA-coding sequences above those 7 already characterized. There is only one single base discrepancy between the two mRNA sequences determined using the dscDNA or the genomic clones. This demonstrates the accuracy and reproducibility of the cloning and sequencing techniques. The ovalbumin mRNA sequence was found to be 1872 nucleotides in length, 13 nucleotides larger than the previous value reported by McReynolds et al. [Nature 273, 723-728 (1978)].
A clone which contains the complete chicken ovalbumin gene, including its leader coding sequences, has been isolated. From electron microscopic analysis of this DNA we conclude that the minimal size of the transcriptional unit for ovalbumin is 7.7 kilobases. The DNA sequence of the region surrounding the 5' end of the ovalbumin gene is presented. Comparison of this sequence with those of other eukaryotic genes reveals striking similarities, possibly related to a promoter region, approximately 30 base pairs upstream from the site coding for the 5' end of the mRNA.
An EcoRI fragment of chicken DNA (fragment 'a') containing a sequence complementary to the 3' half of ovalbumin mRNA has been isolated by molecular cloning. Analysis of the cloned fragment proves conclusively that the chicken ovalbumin gene is split. Fragment 'a' contains no extensive sequence repeated elsewhere in the genome and represents the only type of organisation of this part of the split ovalbumin gene in chicken genome.
The interruptions in the chicken ovalbumin gene which were reported previously (Breathnach, Mandel and Chambon, 1977) are shown to be due to the presence of intervening sequences which separate the messenger-coding sequences. We present evidence for an additional interruption of the gene, which, together with those reported earlier and by Garapin et al. (1978b), make a total of six intervening sequences. All of these intervening sequences are located in the DNA region that corresponds to the part of the ov mRNA which codes for amino acids. The seven coding fragments of the split ovalbumin gene are arranged in the same order and relative orientation as in the ovalbumin double-stranded cDNA. All the sequences coding for ov mRNA are contained in a chromosomal DNA region of 6000 bp, which is more than 3 times longer than ov mRNA. The general organization of the ovalbumin split gene is discussed.
Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ov-mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at exon-intron boundaries, the nucleotide sequence alone cannot define unique excision-ligation points for the processing of a possible ov-mRNA precursor. However, the sequences in these boundary regions share common features; this leads to the proposal that there are, in fact, unique excision-ligation points common to all boundaries.
The ovalbumin gene, is split in chicken DNA. Two interruptions in the sequences coding for ovalbumin mRNA have been detected, at least one of them lying in the protein coding sequence. The unexpected gene organisation is present both in oviduct cells highly specialised in ovalbumin synthesis and in erythrocytes.
The isomerization of 3-phospho-D-glycerate and 2-phospho-D-glycerate catalyzed by the cofactor-independent phosphoglycerate mutase from wheat germ (the isolation and crystallization of which is described in the Appendix) has been shown to be intramolecular by two methods. Mass-spectrometric analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[18O]phospho-D-glycerate shows that there is no exchange of labeled phosphoryl group between carbon skeletons in the mutase-catalyzed reaction. Analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[32p]phospho-D-glycerate by a method involving the kinetic discrimination between 2(-2)H and 2(-1)H species using the enolase isotope effect similarly shows that the wheat germ phosphoglycerate mutase mediates an intramolecular transfer of the phosphoryl group.
From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.
The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.