Datta T.N. Pillay

Structure and sequence evolution of three legume chloroplast DNAs

SummaryChloroplast DNAs from soybean (Glycine max), common bean (Phaseolus vulgaris) and mung bean (Vigna radiata) have been compared in overall structure and nucleotide sequence homologies. Detailed restriction maps demonstrate that the soybean and common bean genomes possess the classical large chloroplast DNA inverted repeat, encoding ribosomal RNA genes, as found previously in mung bean (Palmer and Thompson 1981 a). Heterologous filter hybridizations indicate essentially complete colinearity between mung bean and common bean chloroplast DNAs. Although the linear order of sequence elements is also conserved between soybean and mung bean DNAs, two regions of deletions/additions, each totaling almost 5 kilobase pairs in size, have been identified at the ends of the large single copy DNA region.Alignment and comparison of restriction maps has allowed calculation of nucleotide sequence divergence values for the three DNAs. Mung bean and soybean chloroplast DNAs differ by an average of 10–13% in nucleotide sequence, while mung bean and common bean are significantly more closely related, differing by only 5–6% in base sequence. Base substitutions are distributed non-randomly in these chloroplast DNAs; chloroplast ribosomal DNA is relatively conserved and the two deletion/addition regions relatively diverged in base sequence.

Codon recognition mechanisms in plant chloroplasts

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNAAla(U*GC), cp tRNAPro(U*GG) and cp tRNAArg(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that ‘U:U and U:C wobble’ mechanisms can operate to allow the reading of these codons by cp tRNALeu (UAm7G).

Isolation of flower-specific cDNA clones from sunflower

Abstract Proteins from flower, leaves and roots of sunflower (Helianthus annuus L.) have been resolved by two-dimensional polyacrylamide gel electrophoresis into distinctive patterns. A careful examination of these patterns reveals that many proteins are present in at least two of the three organs, but thateach of these organs has, in addition, a set of specific proteins. Following this observation we have used differential hybridization screening to isolate flower-specific cDNA clones from a sunflower floral cDNA library. Out of 31 clones originally isolated, 12 were subcloned and analyzed by Southern and Northern hybridization experiments. Four clones (SF1, SF2, SF6 and SF7) were shown to cross-hybridize, indicating that they carry the same (or a closely related) insert. Cross-hybridization was also found between clones SF9 and SF19. Using Northern hybridization with RNAs isolated from the individual floral parts we identified an anther-specific clone (SF18), two shown to hybridize to RNA from pistils, from anthers and, to a lesser extent, from corollae.

Cyclic AMP independent protein kinases from soybean cotyledons (Glycine max, L.)

Abstract Two cAMP-independent protein kinases which phosphorylate casein were isolated from post-ribosomal supernatant of soybean cotyledons. They were referred to as casein kinase I (CK I) and casein kinase II (CK II) based on their elution from DEAE cellulose column and purified to 350- and 285- fold, respectively. SDS-polyacrylamide gel electrophoresis data for CK I indicated that it is a single subunit protein of 39 000 daltons, while CK II was observed to be made up of 3 subunits of 52 000, 37 000 and 35 000 daltons. Both CK I and CK II use casein as substrate preferentially over all other substrates tested and CK II phosphorylates 40 S ribosomal subunits and 40 S ribosomal subunit proteins. Both the enzymes were found to undergo autophosphorylation in the presence of [γ- 32 P] ATP, phosphorylating CK I and 35 000-dalton protein band of CK II. The enzyme utilized ATP in preference to GTP in phosphotransferase reactions.

Partial characterization of the gene coding for subunit IV of soybean mitochondrial NADH dehydrogenase.

SummaryBy using the spinach chloroplast atpE gene (ε-subunit coding gene) as a probe we have isolated, from a soybean mitochondrial DNA library, a sequence containing a 405 base-pairs (bp) open-reading frame (ORF). This ORF, which is unique in the soybean mitochondrial genome, is probably part of an exon of the gene coding for subunit IV of the NADH dehydrogenase complex. The predicted protein shows 42% sequence similarity with the C-terminal region the Aspergillus nidulans NAD4 protein. The gene is split by a class II intron which is larger than 1,950 bp. Transcription analysis revealed a single 2,200 nucleotide long transcript which does not contain the intron sequence.

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

SummaryWe have isolated and sequenced an anther-specific gene from sunflower which encodes an 800-nucleotide transcript detectable in the peripheral anther cells. It contains an intron of 2615 bp, which separates the first exon (77 bp) coding for a putative signal peptide of 21 amino acids, from the second exon (563 bp) coding for a 100 amino acid polypeptide. The 5′ and 3′ untranslated regions comprise respectively 13 and 264 bp. The SF2 gene is present in the sunflower genome in several copies, all or most of which contain a closely related intron.

Presence of a chloroplast-like elongator tRNAMet gene in the mitochondrial genomes of soybean and Arabidopsis thaliana

SummaryThe nucleotide sequence of elongator tRNAMet genes from soybean chloroplast and mitochondria and Arabidopsis thaliana mitochondria have been determined. The mitochondrial tRNAMet genes from soybean and A. thaliana are identical, and they differ from the soybean chloroplast tRNAMet gene by only four nucleotides. Analysis of the flanking regions indicates that the mitochondrial tRNAMet gene is not present on a large chloroplast DNA insertion in the mitochondrial genome, but it suggests that they have a common origin. Comparison of the three genes and the evolutionary implications are discussed.

Characterization of Glycine max cytoplasmic, chloroplastic and mitochondrial tRNAs and synthetases for phenylalanine, tryptophan and tyrosine

Abstract Transfer RNAs and aminoacyl-tRNA synthetases of phenylalanine, tyrosine and tryptophan were localized in the cytoplasm, the chloroplasts and in the mitochondria. The aminoacyl-tRNA synthetases of these three aromatic amino acids fractionated by hydroxylapatite chromatography from both chloroplasts and mitochondria were able to aminoacylate Escherichia coli tRNA suggesting their prokaryotic nature. On the other hand, cytoplasmic enzymes were unable to aminoacylate E. coli tRNA and they were found to be distinct from the organellar aminoacyl-tRNA synthetases in chromatographic behaviour as well as in their kinetic properties. Three isoacceptors for each of tRNAPhe and tRNATrp were resolved by reversed phase chromatography (RPC-5) and one isoacceptor was localized in each for cytoplasm, chloroplasts and mitochondria. tRNATyr was resolved into 5 isoacceptors; 2 were localized in cytoplasm, 2 in chloroplasts and the remaining 1 in mitochondria. Organellar tRNAs in all the three cases could be aminoacylated by E. coli aminoacyl-tRNA synthetases while cytoplasmic tRNAs were not aminoacylatable by E. coli enzymes. Codon recognition studies with tRNATyr isoacceptors indicated that all the organellar isoacceptors recognize UAC codon whereas one of the cytoplasmic isoacceptors recognizes UAC codon and the other recognized UAU codon.

Preparation of a tRNA-dependent wheat germ protein-synthesizing system.

A method is described for the preparation of a tRNA-dependent wheat germ protein-synthesizing system. This system can be supplied with exogenous tRNAs of eukaryotic or prokaryotic origin. In order to obtain maximal aminoacylation of the added tRNAs, the translation assays can be supplemented with homologous aminoacyl-tRNA synthetase preparations. Such a tRNA-dependent wheat germ protein-synthesizing system, which is easy to prepare, can be used not only to translate plant cytoplasmic mRNAs in the presence of added cytoplasmic tRNAs, but also to determine the translation activity of exogenous tRNAs from various sources in the presence of either natural or in vitro synthesized mRNAs.

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAsPhe which differ in their minor nucleotide content

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNAPhe species, namely tRNAPhe1 and tRNAPhe2. By sequence determination, we show that tRNAPhe2 is identical to the previously sequenced tRNAPhe1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAsPhe vary during leaf development: in etiolated leaves the undermodified tRNAPhe2 only represents 15% of total chloroplast tRNAPhe, during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.