Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori

Summary The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Isolation of a new paramutagenic allele of thesulfurea locus in the tomato cultivar Moneymaker following in vitro culture

A new allele, SC148, of thesulfurea locus inLycopersicon esculentum was detected in a line derived after repeated selfing of plants that had been regenerated from tissue culture. Like the originalsulf mutant, SC148 displayed two mutant phenotypes: green-yellow speckled plants in which thesulf ^vag allele is present and pure yellow plants homozygous for thesulf ^tpura allele. Although the mutant alleles are recessive to wild-type, an unpredictable number of variegated and pura plants appeared in F₁ progenies that had been derived from crosses between SC148 and wild-type tomato plants. The presence of the wild-typesulf ⁺ allele in these variegated heterozygotes was demonstrated using a cytological marker that is linked tosulf. It is concluded that the mutantsulf allele of SC148, imposes its variegated expression state on the wild-typesulf ⁺ allele present insulf ⁺/sulf^vag heterozygotes. This behaviour, known as paramutation, has also been described for the originalsulf allele. The SC148 allele, however, seems to induce changes at an earlier stage in development. The analogy of this paramutagenic system to dominant position effect variegation inDrosophila is discussed.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms. Communicated by B. J. Kilbey     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

渗透剂对白刺花体细胞胚成熟及萌发的影响

该研究以蔗糖、麦芽糖、山梨醇及PEG(6000)为渗透剂,探讨了不同渗透剂对白刺花体细胞胚发育、胚成熟及萌发的影响。结果表明:白刺花下胚轴形成的胚性愈伤组织接种至MS+2,4-D 0.2 mg·L~(-1)+NAA 1.0 mg·L~(-1)+6-BA 2.0 mg·L~(-1)+TDZ 1.0 mg·L~(-1)+蔗糖40 g·L~(-1)+谷氨酰胺100 mg·L~(-1)+植物凝胶3g·L~(-1)的培养基上,体细胞胚发生率高达66. 21%,总胚数为79个; 7%蔗糖可使体细胞胚成熟率高达64.36%,同时也可提高多子叶畸形胚形成; 2%麦芽糖+2%山梨醇+4%蔗糖组合使体细胞胚成熟率最高达88.89%,畸形胚比例最低; 30 g·L~(-1)PEG培养时,体细胞成熟率最高,为82.35%;鱼雷期的体细胞胚最合适转接,可使体胚萌发率达90.58%,复合糖上培养得到的成熟体细胞胚生根率最高,为87.47%。这为实现白刺花体细胞胚育苗奠定了理论基础,并提供了可行的方案。     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Genetic regulation of theAchaete-scute complex ofDrosophila melanogaster

Summary Mutants in two loci,hairy (h ⁺) andextramacrochaetae (emc ⁺), produce phenotypes corresponding to an excess of function of theachaete-scute complex (AS-C), that is, they cause the appearance of extra chaetae. These mutants, although recessive in normal flies, become dominant in the presence of extra doses of AS-C. Here we study the interactions between these three genes, in an attempt to elucidate their relationships. The results show that the insufficiency produced byh oremc mutants can be titrated by altering the number of copies of AS-C. Moreover, excess of function of AS-C produced by derepression mutants within the complex (Hairy-wing) can also be titrated by altering the number of wild type copies of⁺ oremc ⁺. These specific interactions indicate that bothh ⁺ andemc ⁺ code for repressors of AS-C that interact with theachaete andscute region of the complex respectively.     

Structural and functional importance of theβ-turn in proteins. Studies on proline-containing peptides

We report here two sets of results on proline-containing linear peptides, one of which brings out the role of theβ-turn conformation in the structure of nascent collagen while the other points to the functional importance of the β-turn in calcium-binding proteins. Based on the data on peptides containing the -Pro-Gly-sequence, we had proposed and experimentally verified that theβ-turn conformation in these peptides is a structural requirement for the enzymic hydroxylation of the proline residues in the nascent (unhydroxylated) procollagen molecule. Our recent data, presented here, on the conformation of peptides containing both the -Pro-Gly- and -Gly-Pro-sequences reveal that while theβ-turn in the substrate molecule is required at the catalytic site of prolyl hydroxylase, the polyproline-II structure is necessary for effective binding at the active site of the enzyme. Thus, peptides containing either theβ-turn or the polyproline-II structure alone are found to act only as inhibitors while those with the polyproline-II followed byβ-turn serve as substrates of the enzyme. In another study, we have synthesized the two linear peptides: Boc-Pro-D-Ala-Ala-NHCH₃ and Boc-Pro-Gly-Ala-NHCH₃ each of which adopts, in solution, a structure with two consecutiveβ-turns, as judged from circular dichroism, infrared and nuclear magnetic resonance data. Drastic spectral changes are seen in these peptides on binding to Ca²⁺. Both the peptides show a distinct specificity to Ca²⁺ over Mg²⁺, Na⁺ and Li₊. A conformational change in the peptides occurs on Ca²⁺ binding which brings together the carbonyl groups to coordinate with the metal ion. These results imply a functional role for theβ-turn in Ca²⁺ — binding proteins.     

Giant cell differentiation and lethality of homozygousYellow mouse embryos

The evidence presented herein strongly suggests that lethality of homozygousyellow embryos is expressed at the primary implantation stage of embryonic development. Furthermore the expression of the mutant gene appears to be restricted to a specific cell type—the trophoblast giant cell. The stage of developmental arrest is determined by the degree of differentiation and implantation of these cells. Receptivity of the endometrium to giant cell attachment is also an important contributing factor to lethality of homozygousA ^y embryos.     

Heat shock as inducer of metamorphosis in marine invertebrates

Summary In most sessile marine invertebrates, metamorphosis is dependent on environmental cues. Here we report that heat stress is capable of inducing metamorphosis in the hydroid Hydractinia echinata. The onset of heat-induced metamorphosis is correlated with the appearance of heat-shock proteins. Larvae treated with the metamorphosis-inducing agents Cs⁺ or NH₄ ⁺ also synthesize heat-shock proteins. In heat-shocked larvae, the internal NH₄ ⁺-concentration increases. This fits the hypothesis that methylation plays a central role in control of metamorphosis. In the tunicate Ciona intestinalis, a heat shock is able to induce metamorphosis too. Offprint requests to: M. Walther     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

cell clones and pattern formation: Studies onsevenless,a mutant ofDrosophila melanogaster

Summary sev ^LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev ⁺ mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev ⁺, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev ⁺ seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.     

Bristle patterns and clones along a compartment border in the anterior wing margin ofDrosophila hydei

Summary The bristle pattern along the first longitudinal vein of the wing ofD. hydei differs from that ofD. melanogaster. Instead of a triple row,D. hydei and some allied species show a pattern of five parallel bristle rows of which the medial row (MR) is comparable to the medial triple row (MTR) ofD. melanogaster. Cells of the MR can be made homozygousyellow (y) by induction of mitotic recombination in heterozygousy/y ⁺ females. Until 70 h after egg laying (AEL), the MR clones inD. hydei overlap with one or more of the accompanying dorsal and ventral bristle rows. Between 70 and 120 h AEL the MR clones only overlap with dorsal bristle rows. Some time later they also become separated from both dorsal rows. The resulting MR clone pattern fits with the overall longitudinal clone pattern in the wing blade ofD. melanogaster described by Bryant (1970) and others. The MR clones inD. hydei, however, often show a fragmented appearance with many indentations of the surroundingy ⁺ tissue even when induced after fixation of the DV compartment boundary. This result contrasts with the commonly held notion, derived from work withD. melanogaster, that compartment boundaries are smooth lines.     

The behavioural and physiological strategies of bird and reptile embryos in response to unpredictable variation in nest temperature

Temperature profoundly affects the rate and trajectory of embryonic development, and thermal extremes can be fatal. In viviparous species, maternal behaviour and physiology can buffer the embryo from thermal fluctuations; but in oviparous animals (like most reptiles and all birds), an embryo is likely to encounter unpredictable periods when incubation temperatures are unfavourable. Thus, we might expect natural selection to have favoured traits that enable embryos to maintain development despite those fluctuations. Our review of recent research identifies three main routes that embryos use in this way. Extreme temperatures (i) can be avoided (e.g. by accelerating hatching, by moving within the egg, by cooling the egg by enhanced rates of evaporation, or by hysteresis in rates of heating versus cooling); (ii) can be tolerated (e.g. by entering diapause, by producing heat‐shock proteins, or by changing oxygen use); or (iii) the embryo can adjust its physiology and/or developmental trajectory in ways that reduce the fitness penalties of unfavourable thermal conditions (e.g. by acclimating, by exploiting brief windows of favourable conditions, or by producing the hatchling phenotype best suited to those incubation conditions). Embryos are not simply passive victims of ambient conditions. Like free‐living stages of the life cycle, embryos exhibit behavioural and physiological plasticity that enables them to deal with unpredictable abiotic challenges.     

Differential response to gene dosage experiments involving the two loci which control xanthine dehydrogenase ofDrosophila melanogaster

Summary Assays of xanthine dehydrogenase in flies heterozygous forry indicate that these heterozygotes have about 50–70% of the activity of normal. Thus, thery mutants appear to be similar to other genes in which the heterozygote has lower enzyme activity than the homozygous wild-type. On the other hand, the data obtained fromma-l ⁺ /ma-l heterozygotes indicate no dosage response toma-l. This is substantiated by the insertion ofma-l ⁺-bearing chromosomal fragments into various diploid combinations ofma-l ⁺ andma-l. The results demonstrate that one dose ofma-l ⁺ produces normal amounts of xanthine dehydrogenase activity, even in the presence ofma-l.With 1 Figure in the TextThis work was supported by a grant (RG-8202) from the National Institutes of Health.