Showing papers on "Exon published in 1980"

Exons encode functional and structural units of chicken lysozyme

Abstract: The nucleotide sequence was determined for the chicken egg white lysozyme mRNA and for the exons of the gene together with their flanking intron regions. The exon pattern is to some degree related to the structural subdivision of the final protein product. However, the relationship of exons to functional units of the enzyme is better established. Exon 2 codes for amino acids 28-82, which include the catalytically active residues and a cluster of amino acids which bind rings C, D, E, and F of the oligosaccharide substrate. Exon 3 codes for amino acids 82-108, which give additional substrate specificity, determine the cleavage frame for the alternating N-acetylglucosamine/N-acetylmuramic acid chain, and increase the catalytic efficiency of the active center. Exons 1 and 4, respectively, code for translational signal sequences on the mRNA, for the signal peptide of prelysozyme, and for the amino- and carboxy-terminal regions of the enzyme. These regions increase the stability of the molecule but are not directly involved in the catalytic function.

Nucleotide sequence of the EcoRI E fragment of adenovirus 2 genome

Abstract: The entire nucleotide sequence of the Ad.2 EcoRI E fragment has been determined using the Maxam and Gilbert method. This sequence of 2222 bp, which maps between coordinate 83.4 and 89.7 contains information relative to the early 3 region and to the fiber gene. Altogether with fragment EcoRI D which has been recently sequenced, they cover the entire Early 3 region in which several mRNA were mapped. The aminoacid sequence of the 16K and 14K protein is deduced. The localization of the 14.5K mRNA directing the synthesis of the third E3 known protein is discussed, as well as the hypothetical existence of three other early 3 proteins, which would have a molecular weight of 11K. The initiator ATG triplet of the fiber protein has been found at coordinate 86.1, it is followed up to the end of the fragment by an open reading frame allowing deduction of 80% of the aminoacid sequence of this protein. Sequences known to be frequently present at the border of exon sequence were used to tentatively localize the additional "Z" late leader.

Mapping of heavy chain genes for mouse immunoglobulins m and d.

Abstract: A single DNA fragment containing both mu and delta immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new lambda phage vector Charon 28. The physical distance between the membrane terminal exon of mu and the first domain of delta is 2466 base pairs, with delta on the 3' side of mu. A single transcript could contain a variable region and both mu and delta constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

A mutant immunoglobulin light chain is formed by aberrant DNA- and RNA-splicing events.

Abstract: A mutant immunoglobulin gene has been formed by an abnormal (non V/J) recombination event such that abnormal RNA splicing is required to form a mutant light chain. The structure of the gene suggests that the small palindrome thought to be involved in V/J joining also provides the basis for this abnormal DNA recombination and that the absence of a J segment and RNA splice signal allows an abnormal RNA splicing reaction to occur.

Directed Deletion of a Yeast Transfer RNA Intervening Sequence

Abstract: Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

Cloning and complete nucleotide sequence of human 5'-alpha-globin gene

Abstract: We have cloned one of the two human alpha-globin genes and report its complete nucleotide sequence. The gene is 832 base pairs (bp) long from the 5'-cap site to the 3'-polyadenylylation site. The amino acid coding sequences are separated into three segments (exons) by two short (117 and 140 bp) intervening sequences. Highly conserved regions are identified in the 5'-flanking region, intron-exon junctions, and 3' noncoding regions that may have functional significance.

Mouse immunoglobulin D: messenger RNA and genomic DNA sequences.

Abstract: The molecular structure of a mouse immunoglobulin D from a plasmacytoma tumor and that of the normal mouse gene coding for immunoglobulin D are presented. The DNA sequence results indicate an unusual structure for the tumor delta chain in two respects: (i) Only two constant (C) region domains, termed C delta 1 and C delta 3 by homology considerations, are found; the two domains are separated by an unusual hinge region C delta H that lacks cysteine residues and thus cannot provide the covalent cross-links between heavy chains typically seen in immunoglobulins. The two domains and hinge are all coded on separate exons. (ii) At the carboxyl end of the delta chain there is a stretch of 26 amino acids that is coded from an exon located 2750 to 4600 base pairs downstream from the rest of the gene. Analogy with immunoglobulin M suggests that this distally coded segment C delta DC may have a membrane-binding function; however, it is only moderately hydrophobic. A fifth potential exon (C delta AC), located adjacent to the 39 (carboxyl) end of C delta 3, could code for a stretch of 49 amino acids. The tumor9s expression of the delta gene may be aberrant, but the simplest interpretation would be that this tumor expresses one of the several biologically significant forms of the delta chain.

Rescue of a splicing defective mutant by insertion of an heterologous intron

Abstract: It is now widely accepted that the primary transcripts of many eukaryotic genes contain intervening sequences (introns)1–3. These introns, which vary considerably in length, have been found in both coding and noncoding regions4–15. They are removed from the primary transcript in one or more steps by a process called RNA splicing16. The role of RNA splicing, the size and position of introns and the signals which modulate the splicing process are under intense investigation. These studies have been greatly facilitated by the use of viral mutants or viral–eukaryotic recombinant molecules. The SV40 system has been of particular value in elucidating certain genetic elements which are critically involved in the splicing process17–21. Further insight into the biological significance of the splicing process has come from a mutant from which precisely one intron has been removed (intron-minus mutant, dl-2350). This deletion mutant has the potential to circumvent the need for splicing. However, the inability of dl-2350 to accumulate late viral transcripts indicates that splicing is a requirement for the biogenesis of stable mRNA22. This finding is supported by studies using the mouse βmaj globin gene inserted into SV40. The inserted portions contained either the complete globin gene23 or a segment encompassing the intron plus the entire 3′ end of the gene24. As polyadenylation is known to precede splicing2,3, the possibility exists that every transcript possesses sequences near the 3′ end that are directly involved in the splicing process. We report here that, to test this possibility as well as to pursue the question of whether the splicing event and/or defined sequences in the intron are required for the biogenesis of mRNAs, we inserted an isolated intron lacking its own genomic 5′ and 3′ ends into the intron-minus mutant previously described. The experimental protocol provided for obtaining both possible orientations of the insert relative to the late genomic region. Investigation of the transcriptional products indicated that stable mRNA was produced only by the mutant containing the intron in the sense orientation.

Mutations within an intron and its flanking sites: patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA.

Abstract: We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome. Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of “large” polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptide p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b. Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25–27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2. Only one mutant in box2 resembles box7 mutants with respect to criterion a), and no box2 or box9 mutants resemble box7 mutants with respect to criterion c); criteria b) and d) appear to apply equally well to mutants in all three clusters. Fingerprint analysis, carried out with polypeptides p56, p42, p35, p34.5, p30, p26, p23, discloses that a) The polypeptides belonging to the same class of mobility exhibit very similar if not identical sequences in various cases. b) These polypeptide classes, except p56, p42 and p26, share considerable sequence homologies with wild type apocytochrome b, perhaps encompassing 50% or more of the wild type sequences. b) Polypeptides belonging to the classes p42 and p26 exhibit less extensive but nevertheless significant homologies with the wild type sequence. c) Sequences in polypeptides belonging to class p56 are virtually indistinguishable from ones in cytochrome oxidase subunit I. The inferences from these findings are 1) one gene can produce a multiplicity of polypeptide products that share a common sequence at the promoter-proximal (N-terminal) portion, but diverge beyond these regions of homology. 2) Both the multiplicity of products in single mutants, and the protein structure found, argue against the divergent segments to be due to frameshift terminations, and instead suggest that the novel products are consequences of mRNA processing defects (excision and/or ligation) at and near intron regions. 3) Mutations at edges and the center of an intron can generate similar polypeptide patterns, i.e. produce analoguous mRNA processing defects. 4) Mutations in exons, at their boundary with introns, can produce polypeptide patterns indistinguishable from those at the neighbouring intron; they diverge and eventually become typically exonlike in mutants localized at increasing distances from the boundary. 5) Taken together these findings argue that pre-mRNA processing extends beyond the boundaries of the intron proper and that certain exonsequences participate in excision and ligation. 6) Accumulated pre-mRNAs, resulting from defects in splicing can be translated. 7) Product p56 is formally analogous to p23, as a faulty but highly conserved partial product of the wild type protein, the former of Cox I (oxi3 gene), the latter of the cob-box gene proper. Therefore both genes may utilize identical RNA processing elements which are affected by box7 mutations. 8) A small amount of product similar to p56 may accumulate even in some wild types but not in others. This observations suggests that in certain nuclear backgrounds RNA processing may be more error-prone than in others.

Intervening sequences in a Dictyostelium gene that encodes a low abundance class mRNA

Abstract: Using S1 nuclease protection experiments and DNA sequencing, we have identified two intervening sequences (introns) within a Dictyostelium gene that codes for a low abundance class mRNA. The two introns are located within the protein coding region of this gene. Both are small (approximately 100 bp) and extremely (approximately 95%) A + T rich. The splice junction sequences are similar to the splice sites in other eukaryotes. Finally, we have shown that these introns are transcribed as part of a higher molecular weight nuclear precursor.

Structural organization of a 17 KB segment of the α2 collagen gene: evaluation by R loop mapping

Abstract: A recombinant phage, SpC3, containing a 17 kb genomic DNA insert representing approximately 60% of the 3' portion of the sheep collagen alpha 2 gene, was evaluated by electron microscopic R loop analysis. A minimum of 17 intervening sequences (introns) and 18 alpha 2 coding sequences (exons) were mapped. With the exception of the 850 base pair exon located at the extreme 3' end of the insert, all exons contained 250 base pairs or less. The total length of all the exons in SpC3 was 3,014 base pairs. The length distribution of the 17 introns ranged from 300 to 1600 base pairs; together, all of the introns comprised 14,070 base pairs of SpC3 DNA. Thus, the DNA region required for coding the interspersed 3 kb of alpha 2 collagen genetic information was 5.6 fold longer than the corresponding alpha 2 mRNA coding sequences.

Yeast mitochondrial intron products required introns for rna splicing

Abstract: With the goal o f i nvestigating the functions of introns we have b een studying the cytochrome b (or box) and oxi-3 genes of yeast mitochondr i a . ' They a re encoded in the 68 to 76 kb mitochondrial DNA (1) a l ong with only f ive othe r known p r oteins and all of the ribos omal and transfer RNA ' s required for mitochondrial protein synthesi s (see Figure 1) . Although several mitochondria l genes (o li-l, oli-2, oxi-2, and nine tRNA's) have been shown by sequencing to be continuous throughout their coding regions (2-8) three genes (box, 19 rRNA, oxi 3) are thought t o be split. The cytochrome b a nd 19 rRNA introns have been located by R-loop electron microscopy, blot hybridi zation and genetic mapping (8-13). The arguments for oxi-3 being sp lit a r e mor e indirect (11 ,14) . For the investigation of split genes this system has a number o f impressive advantages over most o the rs at present: 1) Most mitochondria11y e ncoded RNA's and proteins have been mapped a nd alter ations in thes e macromolecules are particularly easy to detect. 2) Petite deletions provide a s table way to inhibit mitochondrial protein synthesis. 3) Mutations are easily i sol a t ed and characterized by complement ation and fine structure petite deletion mapping. 4) Of about 200 mutations in cytochrome b characterized to date about half affect expression of anot her mitochondrial gene oxi-3. All o f these p leiotr opic cytochrome b mutations affect trans-acting elements (defined by complementation p r operties) and many map to within the introns of the cytoch r ome b gene.