Pollen limitation meets resource allocation: towards a comprehensive methodology

The standard method of measuring pollen limitation is to add pollen to a number of flowers, preferably to a whole plant, and to compare fruit and seed set with that of naturally pollinated flowers on other plants. In 25 yr of research, this method has yielded valuable data, but it is difficult to use in large plants. This has caused a bias in the available data towards smaller, herbaceous plants with relatively few flowers. I argue that, in order to widen our knowledge of how pollen limitation affects plants, we should go beyond whole-plant pollen addition and change our concept of how a flowering plant functions. The traditional method does not take into account the variation in and dynamics of resource allocation and pollen availability. The concept of integrated physiological units (IPUs) does, but, although it has been applied to pollination biology, it has not received the attention it deserves. I use this article to present its merits again, to propose a step-by-step methodology for studying pollen limitation, and to examine factors influencing possible plant strategies.     

The U3 Promoter and the nef Gene of Simian Immunodeficiency Virus (SIV) smmPBj1.9 Do Not Confer Acute Pathogenicity upon SIVagm

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4⁺ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4⁺ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.     

Characterization of two CTR-like protein kinases in Rosa hybrida and their expression during flower senescence and in response to ethylene

The expression of two CTR-gene homologues was investigated during flower senescence in two Rosa hybrida cultivars. A fragment of a gene for a protein kinase, termed RhCTR1 (GenBank Acc. No. AF271206), was amplified by PCR and used to isolate the corresponding full-length cDNA (Acc. No. AY032953) from a rose petal cDNA library. The protein RhCTR1 has 66% amino acid identity to Arabidopsis CTR1. A fragment of a second CTR homologue, termed RhCTR2 (Acc. No. AY029067) is 69% identical to the corresponding region of RhCTR1. RhCTR1 expression increased during flower senescence, while RhCTR2 was constitutively expressed during flower development. The expression of both RhCTR1 and RhCTR2 was increased in response to exogenous ethylene.     

Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.     

Isolation and characterization of nucleoprotein assembly intermediates of tobacco mosaic virus

During assembly of tobacco mosaic virus from pure RNA and 20S capsid protein aggregates under conditions where protein is limiting, partially assembled intermediates of specific sizes accumulate; these were isolated on sucrose density gradients. The earliest intermediate found in substantial quantity sedimented at 56 S and was shown, by measurement of its buoyant density and of the length of the RNA segment protected by the capsid protein from nuclease digestion, to consist of RNA that is 13% encapsidated (corresponding to a rod length of about 39 nm); the next intermediate sediments at 78 S and is 18% encapsidated (corresponding to a rod length of about 54 nm). Studies of the distribution of intermediates at various input ratios of protein/RNA indicated that their accumulation results from decreases in the rate constants for protein binding that are local to specific points in the course of encapsidation. After extensive nuclease digestion, the RNA still associated with the first intermediate was shown to include a portion that is unencapsidated. This segment of the RNA may be a region of stable secondary that confers the nuclease resistance despite the lack of protection by capsid protein. Such RNA secondary structure, if it exists, would also cause the accumulation of intermediates by imposing an energy barrier to subsequent rod elongation.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Underreplication of satellite DNAs in polyploid ovarian tissue of Drosophila virilis

The satellite DNAs of Drosophila virilis have been examined in diploid and polyploid tissues by isopycnic ultracentrifugation and thermal denaturation experiments. Previous work has established that the satellite DNAs are under replicated in the polytene chromosomes of the salivary glands of D. virilis. The results of the present experiments demonstrate that this underreplication also takes place in the ovaries which contain nurse cells and follicle cells. These tissues are polyploid but do not show polytene chromosomes.     

On the composition of the nucleolus with special reference to its filamentous structure

Summary Different staining procedures, various digestion methods and autoradiographic techniques were employed to study the structure and composition of the nucleolus and of the nucleolonema, after unmasking the latter by adenosine treatment. The presence of DNA, RNA, protein and lipid in these structures has been shown. It has been demonstrated that the filamentous structure within the nucleolus — the nucleolonema— has a core of DNA, around which RNA and protein have accumulated. The structure of the nucleolonema suggests that it is in a highly active state, in synthesizing ribosomal RNA and protein.We take the opportunity to express our gratefulness to the Director, Prof. Dr. Hans Lettré, for providing facilities to work in this Institute. We like to thank our other colleagues, particularly Dr. N. Paweletz, for their valuable help during the course of the investigations.