The ribosomal RNA genes from chloroplasts of mustard (Sinapis alba L.): mapping and sequencing of the leader region

Summary The genes coding for rRNAs from mustard chloroplasts were mapped within the inverted repeat regions of intact ctDNA and on ctDNA fragments cloned in pBR322. R-loop analysis and restriction endonuclease mapping show that the genes for 16S rRNA map at distances of 17 kb from the junctions of the repeat regions with the large unique region. The genes for 23S rRNA are located at distances of 2.8 kb from the junctions with the small unique region. Genes for 4.5S and 5S rRNA are located in close proximity to the 23S rRNA genes towards the small unique region. DNA sequencing of portions of the 5 terminal third from the mustard 16S rRNA gene shows 96–99% homology with the corresponding regions of the maize, tobacco and spinach chloroplast genes. Sequencing of the region proximal to the 16S rRNA gene reveals the presence of a tRNA^Val gene in nearly the same position and with identical sequence as in maize, tobacco and spinach. Somewhat less but still strong homology is also observed for the tDNA ^Val/16S rDNA intercistronic regions and for the regions upstream of the tRNA^Val gene. However, due to many small and also a few larger deletions and insertions in the leader region, common reading frames coding for homologous peptides larger than 44 amino acids can not be detected; it is therefore unlikely that this region contains a protein coding gene.     

Sequence homology between the 32K dalton and the D2 chloroplast membrane polypeptides of Chlamydomonas reinhardii

Summary The region of the chloroplast genome of Chlamydomonas reinhardii containing the gene of the thylakoid polypeptide D2 (psbD) has been sequenced. A unique open reading frame of 350 codons exists in this region. Because the first ATG is followed 11 codons downstream by a second one, the D2 polypeptide consists of either 339 or 350 amino acids. Comparison of the sequences of D2 and the 32K dalton polypeptides, both of which are associated with photosystem II, reveals partial homology. Although, the overall homology of these two polypeptides is only 27%, they contain several related regions and their hydropathic profiles are strikingly similar. These data suggest that the two polypeptides may have related functions and/or that their genes may have originated from a common ancestor. Alternatively, convergent evolution of these polypeptides may be due to structural constraints in the thylakoid membrane. Limited sequence homology is also observed between the D2 polypeptide and some of the subunits of the reaction centers of photosynthetic bacteria.     

Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide

Summary Particular neurons in the nervous system of the Colorado potato beetle, Leptinotarsa decemlineata, are recognized by antisera against bovine pancreatic polypeptide and FMRFamide. Both antisera react with the same neurons. Solid phase absorptions showed that antiserum against bovine pancreatic polypeptide cross-reacts with FMRFamide, whereas antiserum against FMRFamide cross-reacts with bovine pancreatic polypeptide. Some of the immunoreactive neurons have axons branching extensively within the neuropile, which suggests that the peptide is used as transmitter. In the corpus cardiacum, a neurohaemal organ in insects, numerous immunoreactive axon terminals are present. Here, the peptide material is presumably released as a hormone.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.