Changes in the Activities of Aldolase and of D-Glyceraldehyde-3-Phosphate Dehydrogenase during the Mitotic Cycle in Microspores of Lilium longiflorum

Microspores of Lilium longiflorum were isolated at various stages of development surrounding the mitotic interval and were analyzed for changes in the activities of D-glyceraldehyde-3-phosphate dehydrogenase and aldolase. Fructose 1,6 diphosphate was used as substrate. Activities were measured by the increase in optical density due to the reduction of diphosphopyridine nucleotide. It was found that mitosis occurs during the minimal activity of both aldolase and D-glyceraldehyde-3-phosphate, thus indicating that heightened glycolytic capacity is not necessarily related to mitosis. It was also found that soluble-SH levels were highest when the enzymes were least active. It appeared, therefore, that the "—SH enzymes" are not necessarily activated intracellularly by high concentrations of soluble thiol. These results are discussed in connection with the theory that soluble-SH compounds stimulate glycolysis and in this way initiate mitosis.     

CSP41, a sequence-specific chloroplast mRNA binding protein, is an endoribonuclease.

Correct 3' processing of chloroplast precursor mRNAs (pre-mRNAs) requires a stem-loop structure within the 3' untranslated region. In spinach, a stable 3' stem-loop-protein complex has been shown to form in vitro between petD pre-mRNA, encoding subunit IV of the cytochrome b6/f complex, and chloroplast proteins. This complex contains three chloroplast stem-loop binding proteins (CSPs), namely, CSP29, CSP41, and CSP55. Here, we report the purification of CSP41 and cloning of the csp41 gene and show that CSP41 is encoded by a single nuclear gene. Characterization of bacterially expressed CSP41 demonstrates that this protein binds specifically to the 3' stem-loop structure and a downstream AU-rich element of petD pre-mRNA and that its binding affinity is enhanced by associating with CSP55. Our data also show that CSP41 has substantial nonspecific endoribonuclease activity. These data suggest that CSP41 could be involved in 3' processing of petD pre-mRNA and/or in RNA degradation. The fact that different reaction conditions favor RNA binding over ribonuclease activities suggests a possible mode of in vivo regulation.     

The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.