Dauer der DNS-Replikation von Eu- und Heterochromatin bei Microtus agrestis

The duration of DNA replication of eu- and heterochromatin in kidney epithelial cell cultures of female Microtus agrestis was determined with combined H³-thymidine pulse labelling and cytophotometric determination of Feulgen DNA. The average duration of the total cell cycle was 23.3 hrs, with a G1 period of 14.6 hrs, S period of 5 hrs, G2 period of 2.7 hrs, and mitosis of 1 hr. The replication time of eu- and heterochromatin was determined by the frequency of the different labelling patterns after pulse labelling. The time sequence of the labelling patterns was ascertained by DNA measurements. During the S period, euchromatin replicates at first alone for 3 hrs (60% of the length of S) and 1 hr (19.3%) together with heterochromatin. During the last hour (20.7%), only heterochromatic regions replicate. The sex chromatin part of the one X chromosome starts synthesis 20 minutes (7.3% of S) before the remainder of the heterochromatic X material and ends 30 minutes (9.7% of S) prior to the termination of the S period. Replication of euchromatin takes about 80% of the duration of the total S period, whereas that of heterochromatin takes only 40%.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungs-Gemein-schaft.     

DNS-Reduplikationsmuster bei den Riesen-Geschlechtschromosomen von Microtus agrestis

Summary The X-chromosome of Microtus agrestis (2 n=50), comprising about 20 per cent of the homogametic haploid (AX) set, is the largest X-chromosome reported so far in placental mammals. It is four times the size of the X possessed by a great majority of mammals, including the human and the mouse. The Y-chromosome is also enormous, almost three-fifths the size of the X.The present cytological study concerned somatic interphase and prophase nuclei as well as the DNA replication pattern revealed by labeling cultured bone marrow cells with tritiated thymidine.In the male nuclus, the entire Y as well as the long arm and proximal part of the short arm of the X are late labeling and positively heteropycnotic. In the female, one entire X is late labeling and condensed, while the other X shows the same labeling pattern as the male X. Thus the pattern of inactivation of this huge X is such that in each diploid nucleus of both sexes, the amount of euchromatic X-chromosome material is the same as it is in the majority of placental mammals in which the X comprises about five per cent of the haploid set.

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Wesentliche Teile der vorliegenden Arbeit werden von Gertraud Flinspach als Dissertation der Medizinischen Fakultät der Universität Freiburg i.Br. vorgelegt.     

DNS-Replikation und Morphologie der X-Chromosomen während der Syntheseperiode bei Microtus agrestis

X-chromosome behaviour of female Microtus agrestis in interphase nuclei with and without large chromocenters was examined in cultured epithelial and fibroblast cells. By means of pulse-labeling, the configuration of the X-chromosomes in these nuclei can be illustrated; staining with pararosaniline-methylgreen seems to be most suitable. According to the replication behaviour, three types of chromatin can be discerned in the X-chromosomes: early replicating euchromatin, late replicating sex chromatin, and very late replicating heterochromatin. In fibroblasts only the sex chromatin forms a single, small chromocenter; in epithelial cells both the sex chromatin and the remaining heterochromatin form large chromocenters. Both types of heterochromatin replicate their DNA in the condensed state. It seems likely that the late replicating segments of the X-chromosomes (sex chromatin and remaining heterochromatin) are condensed in every cell, but they may not always be configurated or even visible as typical chromocenters; these segments could be distributed over a wide range of compact to more or less diffuse superstructures.     

Translokation und biochemisches Verhalten von D- und L-Phenylalanin bei Vicia faba

Summary D,L-Phenylalanine (phe) applied to the first primary leaf of a young Vicia faba plant moves into the sieve tubes and appears in the honey dew of aphids feeding on the third primary leaf. Ethanol extracts of the treated leaf contain labeled phe and an acidic compound which could be identified as N-malonyl-D-phe.D-phe-l-¹⁴C was obtained pure by enzymatic decarboxylation of the L-isomer of commercially available D,L-phe-l-¹⁴C, using an enzyme from red algae (Hartmann, 1967). The uptake of the D-isomer of phe by the sieve tubes is independent of the age of the treated leaf. L-phe applied to a young leaf is completely incorporated into protein; L-phe taken up by an older leaf is translocated in considerable amounts.Pulse-labeling with the two isomers shows that D-phe entering the sieve tube system is quickly removed to the parenchyma where it is acylated with malonic acid to the phloem immobile N-malonyl-D-phe. L-phe does not react with malonic acid at all. It is translocated to the centers of protein synthesis.

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Herrn Prof. Dr. Maximilian Steiner zum 65. Geburtstag gewidmet.     

Struktur und Position der heterochromatischen Chromosomen in Interphasekernen von Microtus agrestis

Zusammenfassung In Gehirnzellen und Nierenepithelkulturen der Erdmaus, Microtus agrestis, wurden Struktur und Position der Interphasechromozentren untersucht. Die Chromozentren, die von den heterochromatischen Abschnitten der Riesen-Geschlechtschromosomen gebildet werden, erscheinen in Nervenzellen kompakt, in Nierenepithelkulturzellen von unterschiedlichem Kondensationszustand, der vom Zellzyklus unabhängig ist.In Gehirnausstrichen finden sich in 2/3 der Kerne 2 Chromozentren, in 1/3 ein einziges, doppelt großes Chromozentrum, das durch Fusion entstanden ist. Untersuchungen an in Orcein-Essigsäure suspendierten Gehirnzellen zeigen, daß in 88% der Kerne mit fusionierten Chromozentren das Chromozentrum an der Kernmembran, in 12% frei im Kerninneren liegt. In Kernen mit 2 isolierten Chromozentren liegen diese immer an der Kernmembran.Messungen des Abstandes der (in diploiden Kernen) 2 Chromozentren ergeben, daß die Chromozentren 2 Positionen bevorzugen: die Fusion zu einem doppelt großen Chromozentrum und die genau gegenüberliegende Position. Die Verteilung der Abstände deutet auf die Möglichkeit hin, daß sich zumindest die heterochromatischen Chromosomen von einem gewissen Schwellenabstand an somatisch paaren.Eine völlige Übereinstimmung der Position der Chromozentren in Schwesterkernen läßt sich sowohl bei einkernigen getrennten Schwesterzellen als auch in zweikernigen Zellen, die aus einer einkernigen Mutterzelle hervorgegangen sind, beobachten.

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Structure and position of heterochromatic chromosomes in interphase nuclei of Microtus agrestis

Summary Structure and position of chromocenters in interphase nuclei of Microtus agrestis (brain tissue and cultured kidney epithelial cells) were investigated. The chromocenters derived from heterochromatic portions of the very large sex chromosomes appear to be compact in nerve cells and variably condensed in cultured kidney epithelial cells. This variation in condensation is independent of the cell cycle.In smear preparations of brain tissue two thirds of the nuclei contain two chromocenters, whereas one third of the nuclei show one double sized chromocenter, presumably a fusion of two. Suspensions of nerve cells in orcein-acetic acid show that the large fused chromocenters are attached to the nuclear membrane in 88% of the nuclei with one chromocenter. If two isolated chromocenters are present they are always localized adjacent to the nuclear membrane.Measurements of the distance between the two chromocenters show a prevalence of two types of positions: a fusion to a double sized chromocenter and a vis-a-vis position. The frequency distribution of the distances suggest a tendency of somatic pairing of the heterochromatic chromosomes if they come close enough together.In mononucleated sister cells and in sister nuclei of binucleated cells derived from a mononucleated mother cell the position of the chromocenters is identical in the two nuclei.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Heterochromatin,repetitive DNS und Nukleolen in Leberzellen von Microtus agrestis

Zusammenfassung Die Zellstruktur von Leberzellen der Erdmaus, Microtus agrestis, wurde nach Giemsafärbung, Feulgenbehandlung, Behandlung mit Ribonuklease und nach Färbung des konstitutiven Heterochromatins untersucht. Das konstitutive Heterochromatin ist in Leberzellen nicht heteropyknotisch, das fakultative Heterochromatin ist im weiblichen Geschlecht als Sexchromatinkörperchen sichtbar. Bestimmungen des relativen DNS-Gehalts ergaben, daß die Zahl der Sexchromatinkörperchen der Ploidie der Zellkerne proportional ist. Die Nukleolen liegen in Hepatozyten oft randständig; in 59% der diploiden Zellkerne sind 2 Nukleolen enthalten. Nach Anfärbung der repetitiven DNS werden oft auch die Nukleolen gefärbt, nach Ribonukleasebehandlung tritt dieser Effekt nicht auf. Das konstitutive Heterochromatin wird in Form von 2 langen fädigen Strukturen sichtbar.

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Heterochromatin, repetitive DNA and nucleoli in liver cells of Microtus agrestis

Summary The nuclear structure of parenchymal liver cells of embryo and adult Microtus agrestis was studied in smear and section preparations after staining with Giemsa solution and treatment according to Feulgen, after treatment with ribonuclease and after specific staining of constitutive heterochromatin. In liver cell nuclei only the facultative heterochromatin is heteropycnotic, a sex chromatin body is observable in female but not in male animals. Constitutive heterochromatin is not heteropycnotic in liver cells. Measurements of the relative DNA content showed that nuclei with one sex chromatin body are diploid; tetraploid nuclei possess two and octoploid nuclei four sex chromatin bodies. Solely in the diploid cell nuclei of the intrahepatic gall ducts two large chromocenters are found. The nucleoli in hepatocytes often lie at the perimeter of the nucleus. 17% of the diploid nuclei contain one nucleolus, 59% two nucleoli, 23% three and 1% four. After staining of repetitive DNA, the nucleoli often become stained as well; after treatment with ribonuclease this effect does not occur. The constitutive heterochromatin becomes visible in form of two long, threadlike structures. After longer periods of dissociation the sex chromatin body ceases to be visible. Sex chromatin and constitutive heterochromatin are contiguous to the nucleoli.

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Mit dankenswerter Unterstützung durch das Bundesministerium für Bildung und Wissenschaft der Bundesrepublik Deutschland.     

Cytological variations in the constitutive heterochromatin of Microtus agrestis

In the field vole, Microtus agrestis, most of the constitutive heterochromatin is localized in the giant chromosomes. A detailed examination of a large number of adult cell types reveals that this chromatin is actually present as a heterochromatic fiber in all interphase nuclei. Depending upon the cell types, however, the fiber shows varying degrees of condensation and folding ranging from a very long and extended fiber to a large compact chromocenter. The number of cell types with giant chromocenters was less commonly observed than those with extended fibers. This explains why some cells were previously thought to be devoid of heterochromatin.—The results of this investigation strongly indicate that constitutive heterochromatin represents a unique nuclear entity.This investigation was supported by NIH funds (Grant No. HD 1962).     

A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

Adult philopatry and dispersal in the field vol Microtus agrestis

Summary Using mark-recapture data, we related the movements of adult field voles to population density, sex ratio and population growth. Dispersal movements (defined as distances larger than 1 home range diameter) were few in both sexes; 4 out of 197 (2.0%) in males and 8 of 316 (2.5%) in females. The distance moved between sequential trapping periods was similar for males and females; the mean being 10.2 m and 9.0 m respectively. Both males and females moved larger distances during the breeding season than during the nonbreeding period. The distance moved between sequential trapping periods showed a strong negative relation to density, i.e. both sexes moved shorter distances at higher densities, but there were no differences between periods of increasing and declining population densities. These results contradict the dispersal predictions of all major hypotheses proposed to explain population fluctuations in small mammals. The dispersal patterns fit a geometric distribution, suggesting that competition is the primary factor determining the dispersal characteristics of this population.     

Summer acclimatization in the short-tailed field vole,Microtus agrestis

We investigated the changes that occurred in basal and noradrenaline-induced metabolic rate, body temperature and body mass in short-tailed field voles,Microtus agrestis, during exposure to naturally increasing photoperiod and ambient temperature. These parameters were first measured in winter-acclimatized voles (n=8) and then in the same voles which had been allowed to seasonally acclimatize to photoperiod and ambient temperature (6 months later). Noradrenaline induced metabolic rate, basal metabolic rate and nonshivering thermogenesis were significantly higher in winter-acclimatized compared to summer-acclimatized voles. There was a significant positive relationship between basal metabolic rate and noradrenaline-induced metabolic rate. Body mass was significantly higher in summer-acclimatized compared to winter-acclimatized voles. There was a significant positive relationship between body mass and noradrenaline-induced metabolic rate in both winter-acclimalized and summer-acclimatized voles; however, there was no relationship between basal metabolic rate and body mass in either seasonal group of voles. Body temperature after measurements of basal metabolic rate was not significantly different in the seasonal cohorts of voles. However, body temperature was significantly higher in winter-acclimatized compared to summer-acclimatized voles after injection of noradrenaline. Previously we have found that a long photoperiod was not a sufficient stimulus to reduce thermogenic capacity in winter-acclimatized voles during cold exposure, since basal metabolic rate increased to compensate for a reduction in regulatory nonshivering thermogenesis. Here we found that a combination of increased ambient temperature and photoperiod did significantly reduce thermogenic capacity in winter-acclimatized voles. This provided evidence that the two aspects of non-shivering thermogenesis, obligatory and regulatory, are stimulated by different exogenous cues. Summer acclimatization in the shorttailed field vole is manifest as a significant decrease in both basal and noradrenaline-induced metabolic rate, combined with a significant increase in body mass.Abbreviations ANCOV A analysis of covariance - BAT brown adipose tissue - BM body mass - BMR basal metabolic rate - NST non-shivering thermogenesis - NA noradrenaline - V the maximum V recorded following mass specific injection of noradrenaline - V the maximum V recorded following mass specific injection of saline - T _a ambient temperature - T _b rectal body temperature - T _1c lower critical temperature - UCP uncoupling protein - V oxygen consumption     

Pattern of repetitive DNA of the chromosomes of Microtus agrestis

The distribution of repetitive DNA in the chromosomes of Microtus agrestis was studied with the method for demonstrating constitutive heterochromatin given by Yunis et al. (1971) and the reassociation technique described by Schnedl (1971). All autosomes can be individually recognized by means of the position of their bands. The euchromatic segment of the X1 chromosome shows the same banding pattern as the corresponding segment of X2 which consists of facultative heterochromatin. The short arms of the Y chromosome are not deeply stained with either method and therefore do not contain noticeable amounts of repetitive DNA. The relative distances between the bands remain constant during chromosome contraction in mitosis.     

Photoperiod regulates leptin sensitivity in field voles, Microtus agrestis

We have previously shown that cold-acclimated (8°C) male field voles (Microtus agrestis) transferred from short (SD, 8:16 h L:D) to long photoperiod (LD, 16:8 h L:D) exhibit increases in body mass, adiposity and food intake. To assess whether these increases were associated with decreased leptin sensitivity, we infused LD and SD voles with physiological doses of murine leptin (or saline) delivered peripherally for 7 days via mini-osmotic pumps. Measurements were made of body mass (weight-reducing effect of leptin), food intake (anorectic effect of leptin) and gene expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) (thermogenic effect of leptin). The SD animals were sensitive to the weight-reducing effects of leptin (mean body mass decrease of 1.2 g over 7 days) and appetite-reducing effect of leptin (mean food intake decrease of 2.5 g over 7 days), whereas LD voles were resistant to the hormone treatment. The switch from a leptin-sensitive to leptin-resistant state appears to act as a desensitisation mechanism that allows voles transferred from SD to LD to ignore elevated leptin levels generated by increased body fat and accumulate adipose tissue without stimulating compensatory changes opposing the weight gain. Neither SD nor LD voles responded to infusion of leptin by changes in BAT UCP1 gene expression, suggesting dissociation of anorectic and thermogenic effects of leptin, possibly related to chronic cold exposure. Our results indicate that cold-acclimated voles show photoperiod-regulated changes in leptin sensitivity and may provide an attractive model for elucidating molecular mechanisms of leptin resistance.