Richard B. Hallick

Proposals for the naming of chloroplast genes

At the conference tm Structure and Functmn of Plant Genomes held Jn Portese. Italy. ,n September 1982, a meetmg was held to discuss tile poss> b ,hty of making a standard nomenclature fi~r naming genes cod,ng fi~r chloroplast proteins and RNAs Those m at tendan~eagreed that such a standard nomenclature would be valuable and nominated a commit tee of two, X ~ Bot tomley from Canberra and R B Halhck from Boulder, to draft a set o fgu ,dehnes These were fi~rmulated and circulated at tile meeting and the response was. m general , f~.tvorable Subsequently. the dra(t proposal was orcu[ated to various Interested part,es getwven September 1982 and Aprl~ 198a,. w::h an mv~tatton to suggest changes or modlflcatmns At tile contkrence on B,osynthes~s uf the Photosynthetic Apparatus Molecular Bmh>gy. Depar tment . and R c g u l a t u m held m Keystone, Colorado m Apr,l , 198:, a second meet ,ng was held to dtscuss all suggested amendments to the nomenclature, and to fi~rmal,ze a draft proposal fi~r publ ,catmn. The present set of gmdehnes was agreed to by all who attended th,s special workshop on chloroplast gene nomenclature Those m attendance were Warwick Bottomley. Don Bourque. Rose Ann Cat toh(o, Marvin Edelman, A A. Gatenby, John Gray, R B Hall ,ok, Lee Mclntosh, Laurens biers, JeffPalmer,

Biosynthesis of chloroplast transfer RNA in a spinach chloroplast transcription system

We have developed a chloroplast in vitro transcription system capable of transcribing tRNA genes (trn) from the spinach and Euglena gracilis chloroplast genomes. The RNA polymerase contained in the chloroplast extract transcribes the spinach chloroplast trnM2, trnV1, and trnl1 loci and the trnV1-trnN1-trnR1-trnL1 cluster in the EcoG fragment of the Euglena chloroplast genome. Restriction enzyme modified templates were used to demonstrate that the tRNA genes are transcribed in vitro. RNA fingerprint analysis confirmed that tRNAMetm, tRNAlle1 and tRNALeu are correctly processed transcripts from the spinach chloroplast trnM2, trnl1, and Euglena trnL1 loci respectively. CCAOH is added to the mature tRNAs in vitro by a 3 nucleotidyl transferase present in the chloroplast extract. Deletion mutants were constructed from the trnM2 locus to evaluate the role of 5 flanking sequences in transcription initiation and processing. DNA sequences between positions -56 to -85 upstream of the trnM2 locus are required for maximal transcription of tRNAMetm, but are not essential for processing. The RNA polymerase involved in chloroplast trn transcription is distinguishable from the RNA polymerase isolated as a DNA-protein complex from spinach chloroplast that is active in rRNA transcription.

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3-intron/5-exon and four 3-exon/5-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.

Transcription of E. coli and Euglena chloroplast tRNA gene clusters and processing of polycistronic transcripts in a Hela cell-free system

The transcription of cloned tRNA genes that are clustered in the Escherichia coli and Euglena gracilis chloroplast genome was studied in a HeLa cell-free extract. RNA polymerase III transcribes the tRNA gene clusters into polycistronic primary transcripts, consisting of pre-tRNAVal-tRNAAsn-tRNAArg and pre-tRNAAsp-tRNATrp transcribed from the Euglena chloroplast and E. coli tRNA locus, respectively. A UV5-lac promoter in the 5-flanking sequence of the E. coli tRNA gene cluster can be removed without effect on transcription initiation frequency. During transcription of the tRNA gene clusters, RNA polymerase III initiates predominantly, if not solely, with the first tRNA gene. Upon removal of the first tRNA gene, RNA polymerase III initiates transcription with the following tRNA gene. The primary transcripts are subsequently processed to mature tRNAs. RNA fingerprint analysis and reincubation experiments were used to establish the steps of tRNA maturation of the pre-tRNAVal-tRNAAsn-tRNAArg. The 3 trailer is processed prior to the removal of 5 leader and before cleavage of the primary transcript in the spacer sequences. The pre-tRNAs are then processed to mature tRNA molecules.

Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system

The trancription of a cloned trnV1-trnN1-trnR1 cluster from Euglena gracilis chloroplast (ct) DNA and the processing of a tRNAVal-tRNAAsn-tRNAArg polycistronic precursor were studied in a spinach ct transcription extract. A soluble ct RNA polymerase selectively transcribes the trnV1-trnN1-trnR1-trnL1 locus in the EcoG fragment from the Euglena ct genome. Restriction enzyme modified templates and RNA fingerprint analysis were used to confirm that the tRNA genes were correctly transcribed. The tRNAVal-tRNAAsn-tRNAArg polycistronic precursor transcribed by RNA polymerase III in a HeLa cell extract was used as a substrate to demonstrate that a ct tRNA precursor molecule is correctly processed by the ct tRNA processing enzymes. The oligonucleotide pattern of tRNAs processed in vitro from the tRNAVal-tRNAAsn-RNAArg polycistronic precursor is indistinguishable from tRNAVal, tRNAAsn and tRNAArg transcribed by the ct RNA polymerase and processed in the ct transcription extract. The 3′-CCAOH is added to the tRNAs by a 3′ nucleotidyltransferase after correct processing of the 3′ terminus. Correct pseudouridylation was demonstrated for uridine residues in a tRNAMetm molecule transcribed from a spinach ct trnM1 locus. Thus, the enzymatic activities involved in tRNA biosynthesis in vitro include DNA-dependent (tDNA) RNA polymerase, a 5′-processing activity (RNase P-like), a 3′-exonuclease, an endoribonuclease involved in 3′-tRNA maturation, a tRNA nucleotidyltransferase, and pseudouridylate synthetase.

Identification and partial DNA sequence of the gene for the α-subunit of the ATP synthase complex of Chlamydomonas reinhardii chloroplasts

Chlamydomonas Chloroplast DNA atpA ATP synthase DNA sequence

Organization and Expression of the Chloroplast Genome of Euglena gracilis

We have been involved in a characterization of the chloroplast (ct) genes of the unicellular photosynthetic eucaryote Euglena gracitis. Previous work from several different laboratories on physical mapping and cloning of this DNA, and on the characterization of the ribosomal RNA (rRNA) operons has been recently reviewed.1 In the present article we describe our recent studies on the organization and expression of transfer RNA (tRNA) genes. Nucleotide sequence data is now available for 18 different species. We also describe a new Euglena chloroplast extract that catalyzes DNA-dependent, selective in vitro transcription of Euglena ct tRNA genes.

CHLOROPLAST DNA OF EUGLENA GRACILIS GENE MAPPING AND SELECTIVE IN VITRO TRANSCRIPTION OF THE RIBOSOMAL RNA REGION

ABSTRACT Purified chloroplast 5S, 16S, and 23S rRNAs and tRNAs were labeled in vitro and hybridized by the Southern method to ctDNA restriction fragments and cloned ctDNA fragments. Transfer RNA coding loci were found scattered throughout the genome, as well as in the rRNA coding region. 5S, 16S and 23S rRNAs and tRNA(s) all hybridized to the same strand of a 5.6 kbp DNA, repeated three times in tandem on the chloroplast genome. The RNA product transcribed in vitro by Euglena chloroplast RNA polymerase in a purified, transcriptionally active complex with ctDNA has been characterized as largely a selective transcript of the rRNA coding region. In preliminary experiments, sequence homology between Euglena chloroplast DNA and a fragment coding for the large subunit of RuDP carboxylase in C. reinhardii has been mapped.

Base composition heterogeneity of Euglena gracilis chloroplast DNA

Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content.

SELECTIVE TRANSCRIPTION OF THE EUGLENA GRACILIS CHLOROPLAST CHROMOSOME IN VITRO

ABSTRACT The in vitro transcript of a purified, transcriptionally active chromosome isolated from chloroplasts of Euglena gracilis has been characterized as to base composition, fraction of the chromosome transcribed, kinetic complexity, and RNA sequence content in comparison to in vivo transcribed chloroplast RNA. The data are consistent with the interpretation that the sequences present in in vitro synthesized RNA are the same as those present in vivo in purified chloroplasts.