Chloroplast tRNA gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA (UCC) and tRNA (UCU)

The nucleotide sequences of tobacco chloroplast genes for tRNA^Gly (UCC) and tRNA^Arg (UCU) have been determined. The tRNA^Gly gene has a 691-base-pair intron located in the D stem while the tRNA^Arg gene does not have any intron. The tRNA^Gly and tRNA^Arg genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA^Gly gene is transcribed as a 900-base precursor RNA molecule. The tRNA^Gly and tRNA^Arg deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA^Gly (UCC) and phage T4 tRNA^Arg (UCU), respectively.     

Internal structure of a mitochondrial intron of Aspergillus nidulans.

The intron of the mitochondrial apocytochrome b gene, cobA, of Aspergillus nidulans has been subjected to sequence analysis. It contains an open reading frame of 957 base pairs contiguous with the preceding exon. Regions of the translated open reading frames of cobA and the third intron of the cob gene in yeast show high amino acid homology. Comparison of the cobA intron with this and other yeast introns indicates that cobA codes for a maturase protein that splices out the intron encoding it and possibly other mitochondrial introns. Two very similar decamer peptides are found in the protein sequences of the cobA intron, four mitochondrial yeast introns, and the yeast mitochondrial sequence reading frame 1 (RF-1) and may be diagnostic of one class of maturase-coding introns. Four short DNA sequences, two of which are in the region defined by box9 and box2 mutations in the cob gene of yeast, are conserved in cobA and certain yeast introns. Comparison with three yeast introns strongly suggests that the first 200 base pairs of the open reading frame of the cobA intron do not code for any amino acids present in the putative maturase protein but are required for splicing or the control of splicing, or both.     

Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

A nuclear gene AB80 has been isolated from a phage λ Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80. The first methionine codon 3′ from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A “TATA” sequence occurs 31 nucleotides 5′ from the cap site. A second TATA sequence is found 7 nucleotides on the 5′ side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within the constituent polypeptides of the light-harvesting chlorophyll a/b-protein complex. The sequence Arg-Lys-Ser-Ala-Thr-Thr-Lys-Lys occurs at, or near, the NH₂-terminus of the mature polypeptide encoded by AB80. This basic peptide is of interest because of its apparent involvement in changes in excitation-energy distribution in chloroplast membranes. Some general similarities, but no extensive sequence homology, is found on comparing the transit sequence for the precursor to the chlorophyll a/b-binding polypeptide with the transit sequences previously determined for the precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase.     

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5′ end and 0.43 kb to the 3′ end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.     

Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase consists of large subunits (LS) and small subunits. In plants, the LS is encoded in chloroplast DNA and the small subunit, in nuclear DNA. In cyanobacteria, both subunits are thought to be encoded in chromosomal DNA because of prokaryotes. The gene for the LS of ribulose-1,5-bisphosphate carboxylase/oxygenase from a cyanobacterium, Anacystis nidulans 6301, has been cloned in pBR322 and subjected to sequence analysis. The coding region contains 1,416 base pairs (472 codons). The deduced amino acid sequence of A. nidulans LS protein shows 80% homology with sequences of maize, spinach, and tobacco LS proteins; the nucleotide sequence of A. nidulans LS gene shows 70% homology with sequences of the plant genes. Between A. nidulans LS and the plant LS proteins there is exact sequence homology around the lysine residue to which the activator CO₂ binds and around the two lysine residues to which ribulose 1,5-bisphosphate binds. The amino acid sequence where the LS binds to the small subunit is also highly conserved. From comparison of the LS proteins of A. nidulans and the three plants, the rate of amino acid substitution is estimated to be 0.25-0.5 × 10^-9 per year per site, which is far below the median value of various types of proteins (1.2 × 10^-9 for hemoglobin α). The LS protein is thus a conserved protein.     

An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum.

The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.     

An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.     

Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene.

Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size, near absence of dispersed middle repetitive DNA, and short life cycle. We have cloned and determined the nucleotide sequence of a single-copy gene from A. thaliana likely to be the gene encoding alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The gene was isolated from a random recombinant library by cross-hybridization with a maize Adh1 gene probe. The DNA sequence contains an open reading frame capable of encoding a polypeptide the same length as maize ADH1 and ADH2 (379 amino acids) and having approximately equal to 80% homology with both maize enzymes. This open reading frame is interrupted by six introns whose positions are conserved with six of the nine intron positions present in both maize genes. The 5' and 3' untranslated regions are, respectively, 58 and 204 base pairs long. Sequences important for eukaryotic gene expression such as the TATA box, polyadenylylation signal, and intron splicesite sequences are found in the expected locations. The gene hybridizes to a specific anaerobically induced RNA in Arabidopsis whose appearance correlates with the anaerobic induction of Arabidopsis ADH protein.     

Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants

A potato inhibitor II gene (IIK) was isolated from a library of potato genes in λ bacteriophage. An 8-kilobase-pair (kbp) insert was identified using a tomato inhibitor II cDNA as a hybridization probe, and a 2.6-kbp fragment containing the gene was subcloned into the plasmid pUC13 and characterized. The nucleotide sequence of the isolated gene exhibited 87% identity with the wound-inducible tomato inhibitor II cDNA sequence. The amino acid sequence of inhibitor IIK, deduced from the potato gene, exhibited 84% identity with the tomato inhibitor II protein. A 1000-bp restriction fragment from the 5′ flanking region of the gene was fused to the open reading frame of the chloramphenicol acetyltransferase (CAT) gene. This fusion was terminated in two ways: (i) with a terminator sequence from the potato inhibitor II gene and (ii) with a terminator from the 6b gene of Ti plasmid pTiA6. These chimeric genes were transferred into tobacco cells via a binary Ti vector system, and transgenic plants were regenerated. The CAT gene was expressed in leaves of transformed plants in response to wounding when fused with the inhibitor IIK promoter and terminator regions. The chimeric gene containing the 6b terminator did not express CAT in response to wounding. The wound-inducible expression of CAT activity was systemic and was induced in tissues distal to the wounded tissues. The time course of wound induction of CAT activity in transgenic tobacco leaves is similar to that found for wound-inducible inhibitor I and II mRNAs in tomato leaves. These results demonstrate that sequences necessary and sufficient for wound inducibility are present within ≈1000 bp of the control regions of the inhibitor IIK genes and that wound-inducible components of tobacco leaf cells can regulate these sequences.     

Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii.

The complete nucleotide sequence (119,707 bp) of the black pine (Pinus thunbergii) chloroplast genome has been determined. It contains 4 rRNA genes and 32 tRNA genes. To our knowledge, the tRNAPro (GGG) gene has not been found in any other chloroplast genome analyzed. Sixty-one genes encoding proteins and 11 conserved open reading frames are also found. Extensive rearrangements are apparent in the chloroplast genome relative to those of other land plants. The most striking feature is the loss of all 11 functional genes (ndh genes) for subunits of a putative NADH dehydrogenase that are found in the chloroplast genomes of angiosperms and a bryophyte. Four ndh genes were completely lost and the other 7 genes remain as obvious pseudogenes. This unexpected finding raises the possibility that all ndh genes have been transferred to the nucleus or that an NADH dehydrogenase is not essential in black pine chloroplasts.     

Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone.

We have detected a variant species of bovine growth hormone mRNA in bovine pituitary tissue and in a stably transfected bovine growth hormone-producing cell line. Analysis of this variant mRNA indicated that the last intervening sequence (intron D) had not been removed by splicing. Inspection of the sequence of intron D reveals an open reading frame through the entire intron, with a termination codon encountered 50 nucleotides into the fifth exon, which is shifted from the normal reading frame in this variant mRNA. If translated, this variant mRNA would encode a growth hormone-related polypeptide having 125 amino-terminal amino acids identical to wild-type growth hormone, followed by 108 carboxyl-terminal amino acids encoded by the 274 bases of intron D along with the first 50 nucleotides of exon 5. This variant polypeptide would be 42 amino acids longer than wild-type bovine growth hormone or approximately 5000 greater in molecular weight. The intron D-containing variant of bovine growth hormone mRNA was demonstrated to exist on polysomes, suggesting that this mRNA species is translated into a polypeptide. Cytosolic mRNA species containing any of the other three introns of the bovine growth hormone gene were not detectable.     

Nucleotide sequence and organization of the mouse adenine phosphoribosyltransferase gene: presence of a coding region common to animal and bacterial phosphoribosyltransferases that has a variable intron/exon arrangement.

We have determined the nucleotide sequence of a functional mouse adenine phosphoribosyltransferase (APRT) gene and its cDNA. The amino acid sequence of the enzyme is deduced from an open reading frame in the cDNA and predicts a protein with a molecular weight of 19,560. The protein coding region of the gene is approximately 2 kilobases, and it is composed of five exons and four introns. While the body of the gene is 53% G + C, the 200 nucleotides upstream from the ATG translation start codon are 66% G + C and contain three copies of the sequence C-C-G-C-C-C. The mouse APRT enzyme shares a homologous 20-amino acid sequence with mouse, hamster, and human hypoxanthine phosphoribosyltransferases (HPRTs) and several bacterial phosphoribosyltransferases. This sequence has previously been shown to be a likely catalytic domain in human HPRT and Escherichia coli glutamine phosphoribosyltransferase. Because of the similarities in function of these proteins, both eukaryotic and prokaryotic, it is not unexpected that they should exhibit one or more regions of homology, particularly at the 5-phosphoribosyl-1-pyrophosphate and purine binding sites, especially if they are related via a common evolutionary lineage. This homologous sequence is interrupted by a single intron in the mouse APRT gene and by two introns in the mouse HPRT gene. Furthermore, the positions of both introns in the HPRT sequence are different from that of the single intron in the corresponding sequence of the APRT gene.     

Genes for the eight ribosomal proteins are clustered on the chloroplast genome of tobacco (Nicotiana tabacum): similarity to the S10 and spc operons of Escherichia coli.

The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined. RNA gel blot hybridization revealed that all eight coding regions are expressed in the chloroplasts. The arrangement of the eight genes resembles that found in the E. coli S10 and spc operons. Among the eight genes, the L2 and L16 genes contain 666- and 1020-base-pair introns, respectively. These intron boundary sequences are consistent with the conserved boundary sequences of the chloroplast group III introns [Shinozaki, K., Deno, H., Sugita, M., Kuramitsu, S. & Sugiura, M. (1986) Mol. Gen. Genet. 202, 1-5].     

Exon/intron organization of the chicken type II procollagen gene: intron size distribution suggests a minimal intron size.

Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH-terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH-terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence.     

Structure and expression of Hox-2.2, a murine homeobox-containing gene.

The Hox-2.2 gene is one of a cluster of homeobox-containing genes on mouse chromosome 11. A cDNA clone containing the Hox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 cDNA and genomic clones indicates that there are at least two oxons and one intron. The largest open reading frame includes the homeobox and codes for a 224 amino acid protein of molecular weight 25,312. Comparisons of the predicted Hox-2.2 protein with other homeodomain-containing proteins revealed four regions of sequence similiarity: an N-terminal octapeptide, a hexapeptide upstream of the homeodomain, the homeodomain, and a glutamic acid-rich region at the C terminus. Possible functions of these regions are discussed. The Hox-2.2 gene is expressed in 13.5-day embryos in the developing hindbrain and spinal cord. The expression patterns of Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared.