Leucine-lysine synchronization of Allomyces germlings

Summary The leucine-lysine synchronization technique of Dill and Fuller (1970) has been further refined and used to study various biosynthetic events of pre-mitotic germlings of Allomyces neo-moniliformis (the time of DNA replication, RNA synthesis, and protein synthesis), and various morphogenetic changes (germling development, nuclear cap breakdown, and the first mitotic nuclear division). The degree of synchrony induced in a population of germlings appears to be determined by the time when the zoospores are induced to encyst and germinate rather than by the duration of the swimming period of the zoospore. DNA replication, nuclear cap breakdown, early protein synthesis, and morphogenetic development appear to occur prior to messenger RNA synthesis in developing thalli and thus would be under the control of pre-existing messenger RNA. The degree of synchrony of particular morphogenetic or biosynthetic developmental changes induced in a population of A. neo-moniliformis germlings must be determined for each aspect of development which is to be studied.     

Colchicine and the mitotic spindle of the aquatic phycomycete Allomyces

Summary Exposure of germlings of Allomyces neo-moniliformis to colchicine for 0 to 5 min after zoospore encystment was found to block 30% of germlings derived from flagellated zoospores and 55% of germlings derived from deflagellated zoospores in C-metaphase configurations at the first mitotic division. The zoospore lacks a pool of colchicine binding protein, and protein synthesis is absent during the time when colchicine first becomes effective in inducing C-metaphase. From these observations it is concluded that the microtubule subunit protein of the spindle apparatus of the first mitotic division to a large extent is derived from the depolymerization of the cytoplasmic microtubules of the zoospore. GTP, Mg²⁺, and ATP were observed to be antagonistic to the action of colchicine in vivo. It is suggested that these compounds may compete with colchicine for binding to the subunit protein in vivo. Germlings derived from flagellated zoospores are appreciably less subject to the action of colchicine in the presence of the antagonistic compounds than are germlings derived from deflagellated zoospores. This differential sensitivity to colchicine is interpreted as reflecting a difference in the quantity of microtubule subunit protein present at the time of exposure to colchicine.     

Development of the Amut Bmut strain of Schizophyllum commune

Summary In search for genetically uniform system for the cytological and biochemical analysis of sexual morphogenesis in Schizophyllum, a dikaryon mimic, the Amut Bmut strain, was studied. The course of nuclear distribution and clamp formation was followed in germlings of the double mutant. It was found that this mutant mimics the interaction between two compatible strains. The developmental sequence leading to dikaryon formation can be precisely timed. Thus, it is suggested that this strain be used for determination of the sequential induction of the enzymatic systems operating in sexual morphogenesis.     

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.     

Reactivation of Ribonucleic Acid and Protein Synthesis During Germination of Blastocladiella Zoospores and the Role of the Ribosomal Nuclear Cap

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.     

The Concept of Messenger RNA and Cytodifferentiation

The messenger RNA hypothesis serves as a concrete model of genefunction which may be of real use to the developmental biologist.The hypothesis posits that the product of a gene is an RNA moleculewith a base sequence complementary to the sequence of nucleotidebases in the DNA. The operations and restrictions which definea messenger are discussed. Animal and embryonic cells, whileconforming in general to the predictions, display certain variationsfrom the original hypothesis, such as difficulties in obtainingdirected synthesis of protein in vitro, greater stabilityofthe messenger RNA, association of active ribosomes with lipoproteinmembranes, and a possible heterogeneity of ribosomes. Some recent studies on messenger RNA in sea urchins and frogembryos are discussed, with special reference to the natureand importance of the RNA synthesized during cleavage, the significanceof new ribosome synthesis, and the potential importance of controlmechanisms at the 'translational level for regulation of newprotein synthesis The latter point is illustrated further by a discussion of theinitiation of synthesis of hemoglobin in the chick embryo. Theapplication of drugs and antimetabolites which derange and inhibitRNA synthesis (actinomycin, 8-azaguanine, 5-fluorouracil, and5-bromodeoxyuridine) shows that 8 hours prior to its onset,the synthesis of hemoglobin is independent of the synthesisof RNA of high molecular weight. The initiation of hemoglobinsynthesis is probably regulated at the translational level.Preliminary experiments using the heme precursor, delta-amino-levulinicacid, show that heme may be limiting for the onset of hemoglobinsynthesis.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.     

Germination of desiccated aged akinetes of alkaliphilic cyanobacteria

Morphological and biochemical changes associated with synchronous germination of mature, aged and desiccated akinetes of two alkaliphilic cyanobacteria, Cyanospira rippkae and Cyanospira capsulata, are described. Akinetes of both strains proved to be highly resistant to desiccation, being able to germinate, in the presence of either N₂ or nitrate as nitrogen source, with a germination frequency of more than 90% after seven years of storage in a dried state. The first cell division occurred after 8–10 h of incubation, thereafter the germlings of the two strains followed a different pattern of cell differentiation. Heterocysts were first noted, in a terminal position, at 16–18 h in three-celled germlings of C. capsulata and at 21–24 h in C. rippkae, when germlings were at least seven cells in length. Akinetes of both species possessed, on a per cell basis, almost identical amounts of all photosynthetic pigments but, under nitrogen fixing conditions, photosynthetic activity (oxygen evolution) was detected only after new proteins had been synthesized, before a functional heterocyst was developed and while total nitrogen remained constant. With energy provided by aerobic respiration, a wide range of intracellular amino acids characteristic of proteins was utilised to sustain the new protein synthesis. The end of this biosynthetic activity coincided with the timing of the first cell division. From this stage on, no changes in protein concentration occurred until mature heterocysts were developed. In the presence of nitrate, no significant changes in the major germination events were observed.     

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.     

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.     

Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage

Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3′ end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10–13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.     

Macromolecular synthesis accompanying the transition from spores to vegetative forms ofStreptomyces granaticolor

The rates of RNA, protein and DNA synthesis were estimated in synchronously germinating spores ofStreptomyces granaticolor. Rapid uptake of labelled precursors of RNA and proteins was observed after 20 s. The germination process took place through a sequence of time + ordered events. RNA synthesis started after 3 min of germination, protein synthesis began at 4 min and net DNA synthesis at 60–70 min of germination. A characteristic feature of germination was the biphasic pattern in the rate of RNA and protein synthesis. Spores ofStreptomyces granaticolor were sensitive to actinomycin D, rifampicin and chloramphenicol even at the start of germination. Protein synthesis during germination was dependent on new mRNA synthesis and was independent during the first 60–70 min on replication of the spore genome.