Marco Crippa

Large scale isolation of nuclei and nucleoli from vitellogenic oocytes of Xenopus laevis

A new method is described which allows the purification of large quantities of nuclei (germinal vesicles) from Xenopus laevis vitellogenic oocytes of different developmental stages. From the isolated nuclei, purified nucleoli were obtained. The isolated germinal vesicles are transcriptionally active showing endogenous RNA polymerase activity as well as a high level of activity with an exogenously added template.

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster

Abstract A new method for separating Drosophila egg chambers into different developmental classes ( Jacobs-Lorena and Crippa, 1977 ) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [3H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.

Specific control of messenger translation in Drosophila oocytes and embryos.

Abstract The distribution of messenger RNA between polysomes and mRNP in oocytes and embryos of Drosophila melanogaster has been studied by in vitro translational analysis. Poly(A)+ RNA was purified from polysomes or mRNA from mature oocytes and young embryos. The messenger populations were translated in vitro and the peptides synthesized were separated by two-dimensional electrophoresis. Analysis of the 2D gel patterns enabled the detection of three peptides coded by messengers present predominantly in the mRNA pools of mature oocytes. When DNA-binding peptides were selected from the in vitro translation products, they showed, after separation by two-dimensional electrophoresis, less than 100 spots. The analysis of the 2D gels indicated that three DNA-binding peptides are coded by messengers present only in the mRNP of the oocytes. These messengers are later found in the polysomal fraction of embryos.

The mouse karyotype in somatic cells cultured in vitro

SummaryAn idiogram of the karyotype of the mouse somatic cells in culture was constructed by arranging the chromosomes according to decreasing order of length. The length of the individual chromosomes was then measured in 25 colchicin-blocked, hypotonic-treated metaphases from male C3H mice.It was shown that the 40 chromosomes of the diploid chromosome set of the mouse, although they appeared to be very similar to one another, can be subdivided into five distinct groups, each including chromosomes of similar length. The X chromosome belongs to the second group, the Y chromosome to the fifth group. It is thus possible to identify the sex also in somatic cells.In order to test the reliability of such a classification, a statistical analysis of the chromosome measurements in 25 mitosis of male C3H mice was carried out. The analysis has shown that the length differences between the chromosomes belonging to different groups are highly significant. A high variability among mitoses is however present.

Inheritance of rDNA spacer length variants in man.

SummaryWe studied the rDNA spacer length polymorphism in a sample of 121 individuals belonging to families of 2–3 generations. Our data, obtained by restriction pattern analysis of genomic DNA, confirmed the limited and discrete nature of this polymorphism. Using the pattern as a genetic marker, we analyzed the segregation of length variants in the different families and we investigated the possible occurrence of unequal crossing-over events among homologous and nonhomologous rDNA clusters. No direct evidence of recombination in the spacer region that we analyzed emeged from our study. All the differences in the restriction patterns observed among individuals from the same family could be explained as resulting from meiotic segregation. Family data showed a multichromosomal distribution of NTS length variants and demonstrated a direct correspondence between the frequency of a variant in the population and its degree of spreading on the different rDNA clusters.

Mass fractionation of Drosophila egg chambers

Abstract Egg chambers from Drosophila ovaries may now be fractionated in large quantities suitable for biochemical work. A suspension of isolated egg chambers is prepared by subjecting ovaries to digestion with 0.067% collagenase. Egg chambers are separated in size classes (which correspond to different developmental stages) by sieving them through a column of superimposed nylon nets. The method can, in principle, be used for the separation of any particles ranging upward from 10 μm in diameter.

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Abstract Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements ( Spohr et al. , 1981 ). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.

Variations in the amount of polysomes in mature oocytes of Drosophila melanogaster

Abstract Quantitative measurements of polysomes and ribosomes of Drosophila melanogaster egg chambers, mature oocytes, and embryos were done using sucrose gradient analysis. The amount of polysomes per egg chamber increases about 20 times from stage 5 to 13, and then remains constant up to the end of embryogenesis. The percentage of ribosomes in polysomes is fairly constant during oogenesis and embryogenesis (56 ± 7%). Depending on the fly population, the percentage of ribosomes in polysomes of mature oocytes varies from 10 to 70%. It is shown that the percentage of polysomes in mature oocytes decreases with the time of retention of the mature oocytes in the ovary. Twenty-four- to thirty-six-hour-old flies kept in optimal conditions retain their mature oocytes for 2–3 hr. These mature oocytes still contain 40–60% ribosomes in polysomes. Conditions are given which allow the obtainment of reproducibly high amounts of polysomes from mature oocytes of Drosophila .

DNA-dependent RNA polymerase C from Xenopus laevis ovaries: Formation of stable heparin-resistant DNA-binding complexes

Despite accumulation of extensive information concerning the properties of the different eukaryotic RNA polymerase classes [ 1 ] , it is not yet clear what kind of molecular interactions are involved in the recognition of a specific gene by these enzymes. The polyanion heparin, as a DNA competitor, efficiently inhibits initiation but not elongation of transcription by either prokaryotic or eukaryotic RNA polymerases [2,3]. Escherichia coli RNA polymerase, upon preincubation with DNA in the absence of nucleotide precursors, is able to form DNA-binding complexes from wnich PNA synthesis can be initiated in presence of heparin with the addition of ribonucleotides [4] . It has also been shown that heparin eliminates all artificial starts in vitro and that only 8 stable resistant complexes are formed on T7 DNA [S] . In contrast to the prokaryotic enzyme, purified RNA polymerase A from rat liver does not form heparinresistant complexes after incubation with various DNA templates [6]. To our knowledge, no attempt to form heparin-resistant complexes with eukaryotic RNA polymerase B has been reported. In this report we present evidence for the formation of stable heparin-resistant DNA-binding complexes by RNA polymerase C from Xenopus laevis ovaries. This process is temperature-dependent, does not require the presence of a divalent cation, and is independent of nicks in the DNA template.

Structural analysis of repetitive sequence elements transcribed in early development of Xenopus laevis

A cDNA library prepared from mRNA ofXenopus laevis embryos was screened with a genomic DNA fragment containing various transcribed repetitive sequence elements.Comparison of the nucleotide sequence of two isolated cDNAs and their genomic relatives allows one to define two transcribed repetitive sequence elements. One of them belongs to a highly reiterated family and consists of a tandem array of homologous subunits of 77–80 bp. The other is reiterated approximately 2 200 times, has a size of 260 bp and displays a conserved region of 135 bp.The data are consistent with the presence of repetitive sequence transcripts in the 3′ part of mRNA molecules.