Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Comparative study ofTriticum aestivum andT. timopheevi genomes using C-banding technique

The somatic chromosomes ofTriticum timopheevi and those of two varieties ofT. aestivum, Chinese Spring and Bezostaya-1, have been identified by a Giemsa staining technique. The data suggest thatT. timopheevi and tetraploid wheats had a common ancestor from which their genomes differentiated due to chromosomal aberrations and the increase of heterochromatin in the chromosomes of theT. timopheevi G-genome. The differences between the chromosomes of the AB and AG genomes result in substitutions and large translocations between these chromosomes in interspecific hybrids.     

Identification of complexes in a spontaneous triploid Rhoeo spathacea

A ring-of-12 chromosomes at meiosis is characteristic of diploid Rhoeo. Each arm has been assigned a letter in accordance with the segmental interchange theory. Adjacent distribution at anaphase I results in nonviable spores while alternate distribution results in only two types of spores, both viable. Each of these two types has six chromosomes which are collectively either an or a complex. The chromosome complement of a diploid contains one of each. A theoretical diakinesis configuration in the spontaneous triploid Rhoeo is a ring with six bivalents alternating with six univalents. Among the twelve connecting positions, Position D is diagnostic in the triploid. If the complex is in duplicate, two short arms and a long arm are connected at Position D, and Univalent cD is attached to two bivalents by their short arms. In contrast, if the complex is in duplicate, two long arms and a short arm are connected at Position D, and Univalent Dd is attached to two bivalents by their long arms. Squashed preparations of PMC stained with acetocarmine were used. Among a larger number of triploid metaphase I cells studied, 53 had identifiable chromosomes. In four of the 53, all 18 chromosomes were identified. Chiasma failures in these four PMC were distributed at random among the 12 positions, and at random relative to arm length. The unique features predicted in the presence of an extra complex were observed. Root tip karyotypes had only four (rather than five) large metacentrics. It is concluded that the chromosome complement of the triploid consists of two complexes and one complex. Implications for the balanced lethal mechanism are discussed.This paper represents part of a dissertation submitted in partial fulfillment of the requirements for a Ph. D. degree in genetics at The Ohio State University, Columbus, Ohio, USA.     

Lethal genes on the sex chromosomes concealed in a population of the mosquito Aedes aegypti L.

A population has been examined in which an overall parity between the sexes hides considerable between-family variation in sex ratio. A proportion of families show highly distorted sex ratios, with either an excess of females or an excess of males. Distorted sex ratios are invariably associated with mortality in the immature stages at a level appropriate to the action of recessive lethal genes. It has been shown that 26% of M-bearing (Y) chromosomes and at least 24% of m-bearing (X) chromosomes carry a recessive lethal gene.Two such genes have been investigated. l kills males and, in a cross between two heterozygotes, gives rise to a sex ratio close to 2:1 (excess families). k kills females and, in a cross between two heterozygotes, gives rise to a sex ratio close to 1:2 (excess families). Selection for excess or excess did not increase the level of sex ratio distortion.No crossing over occurs between k and the M/m locus whereas l shows 5–10% recombination with M/m. A test for allelism confirmed that l and k are not allelic. The penetrance of k is complete whereas l shows somewhat less than full penetrance. The penetrance of l has been improved by selection.The high frequency of lethals remained in the population during the two year period of study. There was evidence for heterosis preserving this frequency, the heterozygotes living longer and producing more progeny. However lethals were no longer to be found after four further years of laboratory culture.     

Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae

Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B (blue or red) and chromatofocusing on polybuffer PBE 94^tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps.The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it desintegrated into assumingly identical smaller subunits (E. coli rom M_r 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). Both forms, dimer and monomer were found catalytically active.Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.Abbreviations APS adenylylsulphate - APS kinase - ATP adenylylsulphate 3-phosphotransferase - DCPIP 2,6-dichlorophenol indophenol - DTT dithiothreitol - GSH reduced glutathione - GSSG oxidized glutathione - HPLC high performance liquid chromatography - -MSH -mercaptoethanol - PAPS 3-phosphoadenylylsulphate - TNBS 2,4,6 tri-nitrobenzenesulphonic acid     

Ultrastructure and division behaviour of dinoflagellate chromosomes

Chromosomes of Prorocentrum triestinum and P. micans have similar substructural and morphometrical values as revealed by electron microscopy of thin sections. However, differences were found between the species in mean length, volume and numerical density of chromosomes, and the volume of the chromosome complement, the nuclear volume and the chromosome number. When examined by a whole-mount procedure both Prorocentrum species have left-handed screw-like chromosomes which end in differentiated telomeres. The chromosomes divide sequentially from one telomere towards the other, presenting a Y and finally a V configuration. At the region where each chromosome divides nascent sister chromatids are connected by two bridges. Sister chromatids have similar quantitative values when compared with each other and with the still undivided chromosome, which suggests that both replication and division take place as coupled events.Supported by CAICYT, grant 2409/83     

Repetitive DNA sequences in Drosophila

The satellite DNAs of Drosophila melanogaster and D. virilis have been examined by isopycnic centrifugation, thermal denaturation, and in situ molecular hybridization. The satellites melt over a narrow temperature range, reassociate rapidly after denaturation, and separate into strands of differing buoyant density in alkaline CsCl. In D. virilis and D. melanogaster the satellites constitute respectively 41% and 8% of the DNA isolated from diploid tissue. The satellites make up only a minute fraction of the DNA isolated from polytene tissue. Complementary RNA synthesized in vitro from the largest satellite of D. virilis hybridized to the centromeric heterochromatin of mitotic chromosomes, although binding to the Y chromosome was low. The same cRNA hybridized primarily to the -heterochromatin in the chromocenter of salivary gland nuclei. The level of hybridization in diploid and polytene nuclei was similar, despite the great difference in total DNA content. The centrifugation and hybridization data imply that the -heterochromatin either does not replicate or replicates only slightly during polytenization. Similar but less extensive data are presented for D. melanogaster. — In D. melanogaster cRNA synthesized from total DNA hybridized to the entire chromocenter (- and -heterochromatin) and less intensely to many bands on the chromosome arms. The X chromosome was more heavily labeled than the autosomes. In D. virilis the X chromosome showed a similar preferential binding of cRNA copied from main peak sequences.—It is concluded that the majority of repetitive sequences in D. virilis and D. melanogaster are located in the - and -heterochromatin. Repetitive sequences constitute only a small percentage of the euchromatin, but they are widely distributed in the chromosomes. During polytenization the -heterochromatin probably does not replicate, but some or all of the repetitive sequences in the -heterochromatin and the euchromatin do replicate.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Properties of components of covariance of inbred relatives and their estimates in a maize population

Summary Properties of three parameterizations, denoted as the C-model, D-model and Q-model, for covariances of inbred relatives under assumptions of no linkage or epistasis are explored and compared. Additive variance in an inbred population with inbreeding coefficient F, ² _AF =(1+F) ² _A where ² _A is additive variance in a panmictic population, if Q-model parameters Q _xx and Q _xy are both zero. Conditions sufficient for this to hold are presented in terms of gene frequencies and dominance contrasts (homozygotes vs. heterozygotes). Some other properties and potential uses of estimates of components in the models are also discussed. Estimates of components in the D-model and Q-model were calculated from a maize (Zea mays L.) study from which estimates of components in the C-model were previously published. Of particular interest were the covariance (Q _xy ) of effects of alleles at complete homozygosity with inbreeding depression effects, the covariance (D ₁) of additive effects at panmixia with inbreeding depression effects and the within-locus variance (D ₂, alias Q _xx ) of inbreeding depression effects. Estimates of Q _xy , D ₁, and D ₂ were small and nonsignificant in most cases. For ear height in the second year of the study, D ₂ appeared to be a major component. In some cases, results were obtained which had contradictory implications (negative D ₂ coupled with positive Q _xy or D ₁, and positive D ₂ coupled with negative ² _D ). A negative estimate of one or the other of ² _D or ² _A was obtained in one of the two within-year analyses for every character. Problems in getting realistic results were thought to be owing to excessive multicollinearity among the coefficients of the components in the expectations of the covariances of the kinds of relatives included in the study. Implications for future studies of this kind are discussed.Journal Article No. 87-3-14 of the Kentucky Agricultural Experiment Station published with the approval of the Director     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes

It is shown by isopycnic density gradient centrifugation that the DNAs of the sibling species Drosophila hydei, Drosophila neohydei and Drosophila pseudoneohydei differ regarding the numbers and proportions of satellite DNA bands. An overwhelming proportion of all repetitive nucleotide sequences of the DNA is contained in these satellite fractions. The majority of the satellites are species specific despite the close phylogenetic and cytological relationship between the three species studied. — By in situ hybridization experiments it is demonstrated that the various satellite sequences occupy different positions within the chromosomes. All types of localization patterns, from a wide spread occurrence in all chromosomes to an apparent restriction to kinetochore regions of single chromosomes, have been observed. Main band DNA, on the other hand, in its hybridization behavior reflects the DNA distribution according to the banding pattern in giant chromosomes. Generally satellite sequences seem to be included in -heterochromatic chromosome regions but no relation to the heterochromatin of the Y-chromosome was found. — Renaturation studies support various evidence that satellite sequences occur in tandemly repetitious units. At least some of this repetitious material seems to be linked to non-satellite DNA sequences or to DNA of other satellites.     

Spermiogenesis,sperm structure and fertilization in the palaeonemertean Cephalothrix rufifrons (Nemertini,Anopla)

Summary The spermatozoan from testes of Cephalothrix rufifrons consists of an elongated, straight head 13–14 m long with a flattened anterior acrosome and a 12.5-m-long nucleus. Placed along one side of the nucleus, is a single tubular 7-m-long mitochondrion. There is no midpiece, but immediately posterior to the nucleus are two centrioles. The tail is at least one and a half times the length of the head. Mature sperm cells were also found in the oviducts of mature females which, combined with the modified structure of the sperm cell, indicates that sperm is transferred during pseudocopulation.Abbreviations A acrosome - C centriole - D gonoduct - DC distal centriole - E epidermis - F flagellum - I intestine - LM longitudinal muscle layer - L lateral nerve - M nitochondrion - MT microtubules - N nucleus - O oocyte - PC proximal centriole - R rhynchocoel - S spermatozoa - SC spermatocyte - SP spermatid     

Effect of cycloheximide on RNA synthesis in Chironomus polytene chromosomes

Modifications in the synthesis of salivary gland RNA were induced by treatments with 10 /ml cycloheximide (CHM) on 4th instar larvae of Chironomus pallidivitattus. After 3, 6 and 24 h CHM treatment, RNA was labeled in vitro, by incubating the salivary glands in a medium containing H³-uridine. The electrophoretical analyses corresponding to the 3 and 6 h treatment showed a stimulation of the non-ribosomal components of the newly synthesized RNA, while preribosomal RNA synthesis appeared depressed. This fact was also confirmed at cytological level, since autoradiograms made after 3 h of CHM treatment showed a reduced H³-uridine label over the nucleolus and an increase of diffuse labeling over the chromosomes. Longer treatments (24 h) causes a considerable inhibition of the synthesis of all RNA species. The role played by protein synthesis inhibition in the aforementioned effects is discussed. — Some of the morphological implications of CHM treatment, such as modifications of the nucleolar structure (nucleolar segregation) are also reported. The use of a squash technique based on glutaraldehyde fixation of the salivary glands, considerably facilitates such studies.     

Induced and developmental variation in chromosomes of meristematic cells

Change in chromosome size in root tip meristems of rye and Allium cepa are induced by growing the plants in solutions differing in phosphorus content. The chromosomes are 50% larger by volume in a high phosphate as compared with a no phosphate solution. Alteration of other elements supplied in culture also induces change in the size of chromosomes. — The size variation is a reflection of change in the chromosome dry mass. In part at least this change in mass is attributable to alteration in the amount of protein. The DNA component of the chromosomes remains unchanged. — A consistent pattern of change in chromosome size, quite independent of that induced by varying the culture solution, is related to age and development. For example, the root tip chromosomes double in size during the first three weeks of growth in rye seedlings. Thereafter the size decreases. As with the induced chromosome changes the protein content alters, the DNA amount remains constant. — Variation in the non-permanent component of the chromosomes in meristems appears to be closely correlated with the rate of cell metabolism.     

The hemoglobins of Artemia salina. V. Genetic variation in hemoglobin 1, hemoglobin 2, and hemoglobin 3

Electrophoretic mobilities of three hemoglobins (Hb1, Hb2, and Hb3) were studied in 15 populations of brine shrimps. Genetic segregation data support the model that Hb2 contains n -polypeptides and n -polypeptides; Hb1 contains 2n -polypeptides. Hb3 contains neither - nor -polypeptides. There is no evidence of linkage of and loci with each other or with the locus (or loci) which governs Hb3 or with the nonhomologous portion of the sex chromosomes. Hemoglobins of different populations may be hybridized in vitro by incubation at high temperature. Reversible dissociation to subunits which contain only one ( or ) polypeptide occurs at 40 C (for Hb1) and at 50 C (for Hb2).Supported by Grant HD-11445 from the National Institutes of Health.     

Differential DNA synthesis in heterochromatic and euchromatic chromosome sets of Planococcus citri

In males of the mealy bug Planococcus citri, Nur (1966) counted five heterochromatic (H) and about 5, 10, 20, 40, or 80 euchromatic (E) chromosomes in testis sheath nuclei which were undergoing endomitosis. He suggested that the H chromosomes were not replicating and that the nuclei were becoming polyploid as a result of successive cycles of replication of only the E chromosomes. This hypothesis was tested using autoradiography with H³-thymidine to detect DNA synthesis and microspectrophotometric measurements of the Feulgen reaction in nuclei to detect quantitative changes in DNA. — The integrated absorbance of the whole nucleus and of the isolated clump of heterochromatic chromosomes (H body) in polyploid testis sheath nuclei were measured using the mechanical scanner of the CYDAC system. The absorbance of the H body was similar in all testis sheath nuclei examined and was not significantly different from the absorbance of a haploid set of H chromosomes measured after meiosis. The absorbance of the euchromatic component varied in different sheath nuclei, the values closely corresponding to the terms of the series 2c, 4c, 8c. This series is expected if the DNA in the E chromosomes is exactly doubled at each cycle of replication. — Autoradiographs showed that most labeled sheath nuclei had silver grains localized exclusively over euchromatin. With one exception, the remainder of the labeled nuclei had silver grains over both euchromatin and the H body. The observation that euchromatin was much more heavily labeled than the H body and that labeled H bodies occurred at a low frequency and only in the presence of labeled euchromatin suggests that the H body did not incorporate the label and that the silver grains over the H body were the result of -particles which originated in proximal euchromatin.     

Die Auslösung kerninduzierter Regeneration bei gealterten kernlosen Zellteilen vonAcetabularia und ihre Auswirkungen auf die Synthese von Ribonucleinsäure und Cytoplasmaproteinen

Summary By grafting a rhizoid with its nucleus onto one end of an enucleate posterior part ofAcetabularia 21 days after enucleation (transplantation —those parts do not show any morphogenetic capacity or net synthesis—or by injecting an isolated nucleus into it (implantation), regeneration occurs at the opposite end of the old enucleate part after a few days: a new stalk, whorls and finally a cap are formed. During the whole process there is a typical difference in morphology and colour of the former enucleate part and the newly formed regenerate. No changes occur in the amount of RNA and soluble cytoplasmic proteins of the former enucleate parts during 20–23 days whereas the growing regenerate shows a linear increase of both substances during the same time; thus it behaves like a normal growing nucleated cell ofAcetabularia. Removal of the nucleus by cutting off the rhizoid from the graft has the same effects as enucleation of normal cells: the net synthesis of RNA in the regenerate ceases, protein synthesis and morphogenetic capacity continue to function for a limited time.

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Identification of a polytene chromosome band containing a male sex determiner of Chironomus thummi thummi

Hybrid males of Chironomus thummi piger x Ch. th. thummi crosses were backcrossed with females of both parental stocks. Fourth-instar larvae of these backcrosses showed sex specific differences in the pairing behavior of region D₃d-g in chromosome arm F of salivary gland chromosome III. — Analysis of the banding pattern of region D₃d-g after RB and quinacrine staining demonstrated that in piger x thummi hybrid males a single selectively stained band occurs within this region in the heterozygous condition at map position D₃e₁. This band could only be found in the thummi chromosome partner, it is heterochromatic and contains AT-rich DNA. In female hybrid larvae, however, such a selectively stained band is present in neither the thummi nor the piger chromosome region D₃d-g. From these results it is concluded that the selectively stained band D₃e₁ represents the male sex determiner of our Ch. th. thummi stock and that the male is the heterogametic sex.     

C-banding pattern and polymorphism of Aegilops caudata and chromosomal constitutions of the amphiploid T. aestivum — Ae. caudata and six derived chromosome addition lines

Summary C-banding patterns were analysed in 19 different accessions of Aegilops caudata (= Ae. markgrafii, = Triticum dichasians) (2n = 14, genomically CC) from Turkey, Greece and the USSR, and a generalized C-banded karyotype was established. Chromosome specific C-bands are present in all C-genome chromosomes, allowing the identification of each of the seven chromosome pairs. While only minor variations in the C-banding pattern was observed within the accessions, a large amount of polymorphic variation was found between different accessions. C-banding analysis was carried out to identify Ae. caudata chromosomes in the amphiploid Triticum aestivum cv Alcedo — Ae. caudata and in six derived chromosome addition lines. The results show that the amphiploid carries the complete Ae. Caudate chromosome complement and that the addition lines I, II, III, IV, V and VIII carry the Ae. caudata chromosome pairs B, C, D, F, E and G, respectively. One of the two SAT chromosome pairs (A) is missing from the set. C-banding patterns of the added Ae. caudata chromosomes are identical to those present in the ancestor species, indicating that these chromosomes are not structurally rearranged. The results are discussed with respect to the homoeologous relationships of the Ae. caudata chromosomes.