An analysis of the technical variables in the production of C bands

Numerous combinations, concentrations, pH's and durations of HCl, NaOH and SSC treatments were tested for the purpose of developing an improved C banding technique for human metaphase chromosomes. Methods of slide preparation, as they affect C banding were also evaluated. — HCl and SSC treatment used separately, for all times and concentrations tested, gave no C banding. All treatment sequences which included an NaOH exposure gave at least some C banding, but also gave considerable swelling and distortion. Surprisingly, the best results were obtained from heat-dried preparations exposed to 0.2 N HCl at 25° C for 15 minutes, no NaOH and subsequently incubated in 2xSSC, pH=7.0 at 62–65° C for 18–24 hours. This technique is now being used routinely, following a G banding technique for homologue identification, to monitor C band variation in human chromosomes. — The pH of the 2xSSC incubation solution was found to be important. Slides treated as above with HCl, but with 2xSSC, pH=6.0 gave only G banding; HCl and 2xSSC, pH=8.0 gave C banding, but considerable chromosome swelling and poor uptake of stain. — Air- or ignition-dried preparations, with the HCl and 2xSSC treatment appeared undertreated and gave a mixture of G and C banding. A brief (30 second) exposure to 0.07 N NaOH between the HCl and 2xSSC steps is recommended. These results are in support of DNA-protein interaction and/or loss rather than denaturation-renaturation as a likely mechanism for C band production.     

Heated giemsa solution for producing more consistent bands on mammalian chromosomes

Summary Highly consistent results were obtained in banding of chromosomes with the 2xSSC method of Sumner et al. modified by the use of preheated Giemsa staining solution at 40–45°C. A 4xSSC method modified in the same way turned out to give the same consistent banding on flame-dried chromosomal specimens. Temperature seems to be a decisive factor in the band-producing capacity of a given Giemsa dye.

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Zusammenfassung Beständig gute Ergebnisse bei der Bandenmusterfärbung der Chromosomen erzielten wir mit der 2xSSC-Methode von Sumner et al., modifiziert durch den Gebrauch von auf 40–45°C erwärmter Giemsalösung. Die auf ähnliche Weise modifizierte 4xSSC-Methode ergab ebenso beständige Bandenmuster bei flammengetrockneten Chromosomenpräparaten. Die Temperatur scheint also ein grundlegender Faktor bei der Bandmusterung auslösenden Fähigkeit einer gegebenen Giemsalösung zu sein.

     

Banding pattern observed in human chromosomes by the modified BSG technique

Summary A successful modification of the BSG technique to reveal C and R bands simultaneously in human chromosomes is described. Conventional air dried preparations were treated first with 0.1 N HCl for 30 min at room temperature, then denatured in freshly prepared 3% aqueous solution of Ba(OH)₂8H₂O for 10 min at 50°C. After rinsing, the slides were incubated for 1 h at 60°C in 2xSSC, and stained with Giemsa. The striking intense staining pattern could be observed in chromosome No. 19.The factors involved in the present technique were analyzed changing the concentrations of the reagents and the treatment time. It was evident that R, T and C bands correspond to a progressive destruction of the chromosome sructure mainly by the Ba(OH)₂8H₂O solution.     

Ribonucleic acid–deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide

Hybridization in 6xSSC (SSC, 0.15m-sodium chloride-0.015m-sodium citrate) at 66 degrees C was compared with hybridization in formamide-6xSSC (1:1, v/v) at 35 degrees C. As expected, the RNA hybridization potential was labile in the former system and stable in the latter. DNA retention by filters was poor in the formamide system, but could be improved. Several other properties of the hybridization reaction were explored and it was concluded that the formamide system is generally superior.     

Small repeated sequences in the chloroplast genome of Chlamydomonas reinhardi

Summary Chloroplast DNA of Chlamydomonas reinhardi contains many inverted repeated sequences. Analysis by hydroxyapatite binding, S1 nuclease digestion, and electron microscopy indicates that these sequences are 0.1–0.3 kilobase pairs in length, are widely distributed in the chloroplast genome, and make up 4–7% of the chloroplast DNA.Abbreviations RNA ribonucleic acid - rRNA ribosomal RNA - RNA complementary RNA - DNA deoxyribonucleic acid - chl DNA chloroplast DNA - HAP hydroxypatite - SSC 0.15 M NaCl, 0.015 M sodium citrate - 0.1xSSC, 2xSSC, 4.67xSSC 0.1, 2, and 4.67 times the concentration of SSC, respectively - TCA trichloroacetic acid - PB NaPO₄ buffer, pH 6.8 - Kb Kilobase - KbP Kilobase pair     

The predatory mite Hypoaspis miles: temperature dependent life table characteristics on a diet of sciarid larvae, Bradysia paupera and B. tritici

Life table characteristics of Hypoaspis miles Berlese (Acarina: Hypoaspidae) fed on a mixture of Bradysia paupera Tuomikoski (Diptera: Sciaridae) and B. tritici Coquillet larvae were investigated in laboratory experiments at 4 temperatures (15, 20, 25, 30 °C) for development time, juvenile mortality, sex ratio, preoviposition period, oviposition period, postoviposition period, age-specific fecundity, and adult longevity. Juvenile development time decreased with increasing temperature from 46 days at 15 °C to 10 days at 30 °C. The lower temperature threshold was 9.9 °C and development required 205 °D. Juvenile mortality decreased from 52% at 15 °C to 3% at 25 °C and then increased to 24% at 30 °C. Preoviposition period varied with temperature from 12 days at 15 °C to 3 days at 25 °C and then increased to about 4 days at 30 °C. Oviposition period decreased with increasing temperature from 58 days at 15 °C to 25 days at 30 °C. The mean number of eggs per female per day increased from 0.4 at 15 °C to 2.3 at 25 °C and decreased to 1.3 at 30 °C. Age-specific fecundity was described by a temperature dependent model from which the maximum daily fecundity rate could be estimated to be attained at 25.6 °C. Female longevity was significantly shorter than for males, and decreased from 90 days at 15 °C to 34 days at 30 °C. Sex ratio was female-biased at all 4 temperatures and increased with temperature up to 25 °C, decreasing at 30 °C. Estimates of net reproductive rate, intrinsic rate of increase, finite rate of increase, mean generation time and doubling time were obtained. The r _m -value increased with temperature from 0.031 day^-1 at 15 °C to 0.133 day^-1 at 25 °C, after which it decreased to 0.112 day^-1 at 30 °C. The study showed that H. miles can develop and reproduce at temperatures between 15 and 30 °C. H. miles and sciarids have approximately the same optimum temperature and thresholds for development and reproduction and H. miles can be used for biological control of sciarids within the temperature range where the pest occurs.     

Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

Effect of temperature on translation of mRNA coding for an extracellular proteinase and cell proteins inBacillus megaterium

The content and the half-life of mRNA coding for the Ca²⁺-dependent metalloproteinase were measured by determining the enzyme activity excreted into the medium by cells pregrown in the absence of Ca²⁺ after addition of Ca²⁺ and actinomycin D. The content of the functional proteinase mRNA was highest at 31°C, which is the optimal temperature for the synthesis of this enzyme. Its half-life was 15 min, 7 min, and less than 2 min at 24°, 35°, and 42°C, respectively. Only the third of mRNA molecules synthesized at 31°C was translated in vivo into an active enzyme at 42°C, when compared with the translation proceeding at 24°C. Two-thirds of mRNA molecules synthesized at 31°C were translated into stable cell proteins at 42°C when compared with translation at 24°C. The mean half-life of mRNAs coding for cell proteins was 6–7 min at 24°C, 3 min at 35°C and 2 min at 42°C.     

Adaptability of carp neutrophilic granulocytes to environmental temperatures: spontaneous cytotoxic activity and cell-adherent activity

This study observed the adaptability of carp neutrophilic granulocytes possessing spontaneous cytotoxic activity to different environmental temperatures. To study the adaptability of neutrophilic granulocytes, two different temperatures (25° C and 10° C) were selected, both for rearing and forin vitroassays, in which the cytotoxicity and the adherent rate against K562 target cells were measured. The cytotoxicity and adherent rate of neutrophilic granulocytes from carp kept at 25° C for 30 days were higher when assayed at 25° C than when assayed at 10° C. On the other hand, in carp acclimated from 25° C to 10° C, the cytotoxicities and adherent rates, when assayed at 25° C, decreased with increasing acclimation times, eventually becoming smaller than the values obtained when assayed at 10° C. After the fish kept at 10° C for a long period were re-acclimated to 25° C, these activities assayed at 25° C again became higher than the activities assayed at 10° C. These results indicated that carp neutrophilic granulocytes adapt their cytotoxic activity and adherent activity to different environmental temperatures. A change in cellular composition in the head kidney was also observed in carp kept at different environmental temperatures. The percentage of neutrophilic granulocytes became higher and lymphocytes became lower in carp that were kept at 10° C for a long period compared with carp that were kept at 25° C for a long period.     

The response of anther culture to culture temperature in Triticum aestivum

Summary The response of anther culture to culture temperature was studied in detail using many varieties, F₁ hybrids and pollen-derived lines of wheat (Triticum aestivum) as materials. The suitable culture temperature for inducing pollen callus (or embryoids) in wheat anther culture ranged from 26 °C to 30 °C, varying with genotypes. But for the great majority of wheat genotypes the suitable culture temperatures lay between 28 °C and 30°C. The most significant genotypic variation in the response to culture temperature was observed in the comparison between the culture at 33 °C for eight days followed by culture at 25 °C (or 26 °C) and the continuous culture at 25 °C (or 26 °C). This genotypic variation in the response to culture temperature is a heritable character which may be controlled by multiple genes. The effect of culture at 30 °C for eight days followed by culture at 26 °C was similar to, or in some cases, better than that of continuous culture at 28 °C, and the effect of culture at 32 °C for eight days followed by culture at 28 °C was similar to that of continuous culture at 30 °C. In the range from 26 °C to 32 °C, the overwhelming majority of pollen calli emerged before the 40th day after anther inoculation, and the higher the culture temperature, the earlier and more concentrated the emerging period of the pollen callus. The pollen callus obtained at high temperatures above 28 °C should be transferred in time onto the regeneration medium at 25°–27°C to induce shoots.     

High temperature enhances ethylene promotion of anther filament growth in Brussels sprouts (Brassica oleracea var. gemmifera)

A study was made of the effect of high temperature on the growth response of Brussels sprout filaments to ethylene. Filaments with or without the anthers attached were incubated continuously at 25 °C or 35 °C for 7 days or for 2 days at 35 °C followed by 5 days at 25 °C. Growth was reduced during both 35 °C treatments compared to that of filaments at continuous 25 °C. Ethylene had little effect on filament growth at continuous 25 °C, whereas with treatment for either 2 or 7 days at 35 °C ethylene promoted filament growth considerably. Thus ethylene effectively overcame the growth inhibition induced by the 35 °C treatment.High temperature treatments reduced ethylene production from filaments alone, and from filaments with anthers attached. The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) and the ethylene action inhibitor AgNO₃ enhanced filament growth at 25 °C but had little or no effect at 35 °C. The relevance of temperature to ethylene sensitivity is discussed in relation to filament growth and to other plant processes in general.     

A new perspective on thermal inactivation kinetics of human serum butyrylcholinesterase

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37°, 40°, 45°, and 50°C for 24 hr. It was observed that the enzyme heated at 45°C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45°C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40°C for 24 hr, the heated enzyme at 45°C for 24 hr could not be reactivated when it was kept at 4°C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45°C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.     

Effect of temperature on growth,reproduction and survival of the freshwater planorbid snail,Gyraulus convexiusculus,vector of echinostomiasis

Studies were carried out to investigate the effects of 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40° and 45°C on growth, sexual maturity, reproduction and survival of the freshwater planorbid snail, Gyraulus convexiusculus, vector of echinostomiasis, under laboratory conditions. The growth rate of juvenile and sexually mature snails was at minimum at 15°C and was maximum at 35°C. Sexual maturation time was minimum at 35°C and maximum at 20°C. Fecundity was minimum at 15°C and maximum at 35°C. The minimum average and maximum number of eggs per egg capsule was reached at 35°C and lowest at 15°C. 30°C was the optimum temperature for survival of juvenile snails, while sexually mature snails reached maximum survival time at 20°C.     

Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender

This study tested whether variable temperatures (from −0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at −0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 °C, 12 °C to 15 °C, respectively). Per cycle pregnancy rate (PC) was measured after one artificial insemination (AI) in uterine body of 200 × 10⁶ total spermatozoa, 7 h (Experiment 1) or 17 h (Experiment 2) before ovulation. In Experiment 1, PC was similar for both conditions (60% (n = 40 cycles) vs. 63% (n = 40), respectively, 5 stallions × 8 cycles). Only velocity VCL and ALH were slightly higher at 15 °C. In Experiment 2, PC was reduced when ambient temperature was low (semen at −0.5 °C to 3 °C; PC = 25%) rather than intermediate (semen at 4 °C to 7 °C; PC = 53%) or high (semen at 8 °C to 10 °C; PC = 50%) (4 stallions × 8 cycles) (P = 0.002). Sperm stored at −0.5 °C to 3 °C had lower acrosome integrity/responsiveness, similar membrane integrity (HOS test) and motilities, and higher VCL and ALH, than semen stored between 4 and 15 °C. These results demonstrate a wide tolerance of equine sperm to variable positive temperatures and air exposure for 22 h storage and more. However, temperatures close to 0 °C are detrimental for fertility.     

High temperature tolerance and heat acclimation of Opuntia bigelovii

Summary The tolerance of Opuntia bigelovii Engelm. (Cactaceae) to high temperature was investigated by subjecting stems to temperatures ranging from 25°C to 65°C for a 1-h period, after which various properties of chlorenchyma cells were examined. The temperatures at which activities depending on membrane integrity decreased by 50% were 60°C for electrolyte leakage, 52°C for staining by neutral red, and 51°C for plasmolysis for plants maintained at day/night air temperatures of 30°C/20°C. Nocturnal acid accumulation, which depends on stomatal opening and enzymatic reactions as well as membrane properties, was half-inactivated at a lower temperature, 46°C. Visual observation indicated that 50% of the stems subjected to a heat treatment of 52°C became necrotic in 2 weeks.Heat acclimation, which is apparently necessary for survival of O. bigelovii in the field, was investigated by raising the day/night air temperatures from 12°C/2°C to 60°C/50°C in 10°C steps every 2 weeks. The heat tolerance of the cellular properties increased with increasing air temperature; for a 10°C temperature increase, the half-inactivation temperature increased 2.9°C for electrolyte leakage, 3.0°C for staining, 3.8° C for stem survival, and fully 6.1°C for nocturnal acid accumulation. The relative order of these four properties with respect to heat tolerance did not change during the hardening, nocturnal acid accumulation remaining the most heat sensitive. The upper temperature for 50% survival was 59° for O. bigelovii when acclimated to day/night air temperatures of 50°C/40°C.     

Bionomics and population growth statistics of apterous virginoparae of woolly apple aphid, Eriosoma lanigerum, at constant temperatures

Development, survival, reproduction and population growth statistics of apterous virginoparae of woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) at constant temperatures of 10, 13, 15, 20, 25, 30 and 32°C are reported. The developmental times of all life stages were inversely related to temperature ranging from 10 to 30°C. Span of total development (time from birth to adulthood) decreased from 57.8 days at 10°C to 11.7 days at 30°C and increased to 16.8 days at 32°C. A good linear model fit (R²>0.96) between developmental rate and temperature in the range 10–25°C was observed for all life stages. The lower developmental threshold was estimated at 5.8°C for instar I, 4.8°C for instar II, 4.9° for instar III and 4.4°C for instar IV. The lower temperature threshold for total development was estimated at 5.2°C. The upper developmental limit was found to be 32°C. Mean degree-day accumulations required for completion of instars I, II, III, IV and total development were: 125.6, 51.0, 47.7, 50.7 and 267.6, respectively. Fecundity, larviposition period and adult longevity were reduced with increasing temperature. Net reproductive rate was greatest at 15°C whereas intrinsic rate of increase peaked at 25°C. Optimal performance, as measured by fecundity, survival and intrinsic rate of increase, ocurred in the range 13–25°C.     

The effects of temperature on the respiration and production of the freshwater nematode Anonchus sp.

Summary Growth and respiration were measured in a species of Anonchus (Nematoda: Plectidae) at 5°C, 10°C, 15°C, 20°C and 25°C. At 5°C no growth was measurable but the organisms remained active. Maximum production occurred at 15°C, but the highest rate of growth occurred at 20°C. Thus, adult size attained is dependent on the temperature of growth. Respiratory energy losses derived from Cartesian diver microrespirometry, increased with temperature up to 25°C. Regression coefficients (b values) derived from a log log linear regression of weight against oxygen consumption varied between 0.574–1.793, the lowest value being attained at 5°C, the highest at 20°C. Based on Q₁₀, production and respiratory energy losses the optimum temperatures for Anonchus appears to lie between 10°C–15°C.     

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase     

Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.     

Nitrogen and carbon mineralization during incubation of two Bangladesh soils in relation to temperature

Summary At temperatures of 20°, 30°, 40°, 50° and 60°C in a Gangetic alluvial soil (G soil, pH 7.6) N-mineralization and nitrification increased with temperature up to 40°C and mineralized N accumulated entirely as nitrate. At 50° and 60°C mineralized N was relatively low and no nitrification occurred. In the Red soil (R soil, pH 5.2) mineralized N increased with temperature up to 40°C, was somewhat less at 50°C and was at a maximum at 60°C. Nitrification was maximum at 30°C but did not occur at 50° and 60°C. In the G soil C-mineralization increased considerably with temperature, whilst in the R soil there were only small differences due to temperature.