Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

A hybrid DNA sequence containing the replication origin of the multicopy yeast plasmid 2 micron circle and an additional repeated sequence can convert maltose-negative into maltose-positive strains

Summary Yeast DNA pools were prepared by ligating partial Sau3A genomic digests from strains carrying various MAL genes into the BamHI site of the yeast-Escherichia coli shuttle vector YRp7. They were used to transform recipient yeast strains that could not utilize maltose since they lacked a classical MAL gene. Transformants were obtained that could use maltose and also formed normal levels of maltase. They were unstable. They would lose the selective marker TRP1 of YRp7 alone, together with the ability to utilize maltose or only the ability to utilize maltose. The insertion of one of the plasmids was used as a hybridization probe for the others and found to share homologous sequences with all. They were then shown to contain the replication origin of the yeast 2 m circle plasmid and additional genomic digests of total yeast DNA. They hybridized at various degrees of efficiency with several bands, indicating that they were part of a family of repeated sequences. Apparently, it was the combination of the replication origin of the 2 m circles with the additional sequences that promoted maltose utilization.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences.

We sequenced the envelope (env) gene and 3' long terminal repeat of a Friend mink cell focus-inducing virus (F-MCFV). We also sequenced the gp70 coding regions for two cDNA clones of another F-MCFV. The deduced amino acid sequence of the env gene products of both F-MCFVs were compared to the corresponding sequences of other MCFVs and of ecotropic viruses. The env polypeptides of the different viruses showed long stretches of homology in the carboxy-terminal half of gp70 and in p15env ("constant region"). The amino-terminal half of gp70 was very similar in all MCFVs, but showed extensive variations relative to the ecotropic viruses ("differential region"). This differential region in all MCFVs is of endogeneous origin. We show evidence that this region carries determinants for ecotropic or polytropic host range. No indication could be found that the env gene products determine the histological type of disease caused by particular MCFVs. When the long terminal repeats of F-MCFV and Friend murine leukemia virus were compared with those of other viruses causing either lymphatic leukemia or erythroleukemia, several nucleotides were localized which might determine the histological type of disease caused by these viruses.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

Electro-fusion of mesophyll protoplasts ofAvena sativa

In order to assay the viability of electrically fused mesophyll protoplasts ofAvena sativa a technique was developed to determine adenylate levels in single protoplasts and fusion products. The results demonstrate that the intracellular ATP/ADP ratios are identical before and after fusion (values between 1.4 and 1.8) and that the time of the rounding up process is directly related to the ATP level of the hybrid. This was shown by the manipulation of the intracellular ATP/ADP ratio in the light using different effectors. Hybrids with an ATP/ADP ratio of 2.3 needed 54 s to round up completely; in the presence of antimycin (inhibition of both oxidative and light-dependent cyclic electron flow: ATP/ADP=1.1) or dibromothymoquinone (plastoquinone antagonist: ATP/ADP=1.0) the time for rounding up was slightly increased (64 s and 76 s respectively), whereas after preincubation with antimycin, dichlorophenyldimethylurea (inhibition of oxidative and light-dependent electron flow) or uncouplers (ATP/ADP=0.19–0.32) this process needed 128–153s for completion. These results are discussed in relation to the viability of electrically induced fusion products and to energy-dependent events involved in the process of fusion.     

Zur Charakterisierung neuronaler und gliöser Elemente im Epithel des Saccus vasculosus von Knochenfischen

Zusammenfassung Das Epithel des Saccus vasculosus des Flußbarsches Perca fluviatilis besteht aus Krönchenzellen, bipolaren Liquorkontaktneuronen (Zahlenverhältnis etwa 41) und Stützzellen. Im Bereich des Saccus kommen Macrophagen vor. Die Krönchenzellen wurden unter verschiedenen Fixierungsbedingungen untersucht. Die Globuli enthalten schlauchförmige Zisternen, die nicht mit den Zisternen des Zellapex in Verbindung stehen. Im Zytoplasma des Zellapex und der Globuli wird bei langdauernder OsO₄-Imprägnation Osmium gebunden. Die Krönchenzellen werden basal von Ausläufern der Stützzellen unterlagert. Sie werden nicht innerviert und entsenden keine Axone.Die bipolaren Neurone sind durch einen im Liquor endigenden Dendriten und ein Axon gekennzeichnet, das in eines der Faserbündel des Epithels eintritt. Der Dendrit trägt 1 bis 2 Zilien. Die Zelle ist reich an Vesikeln und kann am Perikaryon wie an den Ausläufern Synapsen tragen. Im extrazellulären Raum um die Neurone und in vesikulären Strukturen des Apex wird Acetylcholinesterase nachgewiesen.Der Nervus sacci vasculosi dürfte nur afferente Axone von Liquorkontaktneuronen und efferente Fasern, die diese innervieren, enthalten.

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Neuronal and glial cell elements within the epithelium of the Saccus vasculosus in teleosts

Summary The epithelium of the Saccus vasculosus of Perca fluviatilis consists of coronet cells and bipolar liquor contact neurones in a 41 ratio, and supporting cells. The organ also contains macrophages.The coronet cells have been studied after different kinds of fixation. The globules of these cells contain tubelike cisternae, which do not connect with cisternae in the cell's apical protrusion. The cytoplasm of the apical protrusion and to a greater extent of the globules, is stained by longlasting OsO₄-impregnation. The coronet cells have no direct contact with the basement membrane of the organ. They are neither innervated nor have axons.The dendrites of the bipolar nerve cells end with a bulbous structure protruding into the cerebrospinal fluid. The dendrites contain vesicles and each bears 1–2 cilia. The axons join the fiber bundles of the epithelium. There are synaptic contacts on the surface of the neurons and their processes. In some vesicular structures within the apices and more conspicuously within the extracellular space around these cells indications of acetylcholinesterase activity are found.It appears that the nervus sacci vasculosi contains only afferent axons of the bipolar liquor-contact neurons and efferent fibres which form synaptic contacts with these neurons.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.