A method for analysis of chromosomes in hybrid cells employing sequential G-banding and mouse specific C-banding

A sequential banding technique is described for the identification of chromosomes of interspecific hybrid cells with a mouse parent. Metaphases were first G-banded using trypsin-Giemsa to identify individual chromosomes and then the centromeres of the same cells were differentially stained by a C-banding technique specific for mouse chromosomes. This mouse specific C-banding employs treatment with hot formamide-SSC before staining, and the effect of this treatment on the staining of chromosomes from a number of species was investigated. The specific staining of mouse centromeres confirms the parental origin of chromosomes identified by G-banding and allows the rapid recognition of mouse and non-mouse chromosomes in metaphases from many different hybrid combinations.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.     

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure.

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.     

Influence of exogenous DNA on Ypenyl-treated chromosomes ofVicia faba L.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0·01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.     

Changes in elemental composition of human chromosomes during a G-banding (ASG) and a C-banding (BSG) procedure.

Human chromosomes fixed in methanol-acetic acid have been examined by X-ray microanalysis, before, during and after a G-banding and a C-banding procedure. Phosphorus (representing mainly DNA), sulphur and calcium are the most prominent elements in untreated chromosomes. In the G-banding procedure, the calcium is lost during 2 x SSC treatment. In the C-banding procedure, calcium is lost in the preliminary HCl treatment. During the following barium hydroxide treatment a large amount of barium becomes attached to the chromosomes, but is lost again during the subsequent 2 x SSC treatment. In both banding techniques Giemsa staining produces large peaks for sulphur (thiazine dyes) and bromine (eosin), showing that both types of dyes are involved in the staining. Reduction in the phosphorus peak during these procedures may be partly due to extraction of DNA and other chromosomal components, but could also be due to absorption of phosphorus X-rays by heavy elements (barium and bromine).     

An indirect-labeling procedure for the study of specific interactions of proteins with DNA

A semiquantitative and reproducible indirect-labeling procedure for the study of specific protein/DNA interactions using nitrocellulose-filter-immobilized proteins and linear or superhelical DNA molecules is reported. Proteins were immobilized on nitrocellulose filters either by direct dotting or by electrotransfer from polyacrylamide electrophoretic gels. After incubation with the respective DNA (linear restriction fragments or closed circular recombinant DNA plasmids) the paper strips were washed, and specifically bound DNA was denatured by alkali and detected by hybridization with 32P-nick-translated DNA. The quantitation of the reaction was performed by scanning of the autoradiograms of the radioactive spots and determination of the area under the respective peaks. The intensity of the radioactive spots was proportional to the amount of protein present in the dot. The sensitivity of the assay depends primarily on the affinity of the respective DNA to the protein and in the case of mouse liver histone H1AB/mouse alpha-globin gene approximately 50 ng of protein per dot was enough for determination.     

On the distinction between chromosomes No. 21 and No. 22 by the C-banding pattern

Summary Chromosome No. 21 consistently shows a lighter C band at the centromere than chromosome No. 22, allowing reliable differentiation. This distinction was demonstrated in karyotypes with centric fusions t(Dq 21q), trisomy 21 and by comparing the C-and Q-band polymorphism of the short arms of the G chromosomes.

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Zusammenfassung Chromosom 21 weist stets eine schwächere C-Bande am Zentromer auf als Chromosom 22, wodurch diese Chromosomen eindeutig unterscheidbar sind. Belegt wird dieser Befund durch Karyotypen mit zentrischer Fusion t(Dq21q), Trisomie 21, und durch einen Vergleich des C-und Q-Banden-Polymorphismus der kurzen Arme.

     

Chromosome study of Amazona amazonica and A. aestiva (Aves: Psittaciformes): determination of chromosome number and identification of sex chromosomes by C-banding methods

Two parrot species, Amazona amazonica and A. aestiva, submitted to cytogenetic analysis presented a diploid chromosome number of 2n=70 (20M+50m). With the C-banding pattern, the cells of female speciments showed an almost totally heterochromatic W chromosome. No chromosome differences were observed in the two species studied.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos I. Influence of the embryo preparation method

Summary Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode’s solution containing [³H]amino acids (mixture) at a concentration of 27 μCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo “naked”); d) naked embryos cut randomly into pieces (Day 10 mebryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked mebryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first methods (see above) fulfilled the conditions required at the best. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic Analysis of Cell Proteins).     

Processiveness of DNA polymerases. A comparative study using a simple procedure.

In this communication, we describe a simple procedure for analyzing the processiveness of DNA polymerases in general. By choosing conditions for which the number of incorporations per available primer is less than 1, we have reduced the probability of a primer molecule being utilized by the enzyme more than once. The primer-template used was poly(dA)300:oligo(dT)10, and the product was isolated by oligo(dT)-cellulose chromatography. The number of dTMP residues added per association was determined from the [3H]dThd + [3'-3H]dTMP/[3H]dThd ratio of the product after its digestion by micrococcal nuclease and spleen phosphodiesterase. Using this procedure, we have found that Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus alpha- and beta-DNA polymerase are quasi-processive. Most of these enzymes add on the average approximately 10 to 15 nucleotides before dissociating from the template. T5 DNA polymerase, on the other hand, is processive, i.e. it continues to replicate a given template until it is very close to the 5' end of the template. With nicked DNA-like poly(dA):oligo(dT), the processiveness of E. coli DNA polymerase I is increased 2- to 2.5-fold. The significance of this increase in determining the patch size during DNA repair is discussed.     

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.by D. Schweizer     

Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes.

We used mouse-Chinese hamster somatic cell hybrids which lose mouse chromosomes to examine the distribution of murine leukemia virus DNA sequences in the genome of A/HeJ mice. We analyzed total cellular DNA from various hybrid clones for the presence of viral sequences by molecular hybridization and used the Southern blot hybridization procedure to identify viral DNA in cellular restriction endonuclease fragments. Our results show that murine leukemia virus DNA sequences are distributed among many mouse chromosomes in this strain. Chromosome 4 was shown to contain murine leukemia virus DNA sequences.     

Hybridization-based karyotyping of mouse chromosomes: hybridization-bands.

We have developed a method, which we have named hybridization-banding, to identify simultaneously all chromosomes in a mouse metaphase spread. The method uses a combination of hybridization probes labeled with a single fluor to yield a simple, unique, readily identifiable hybridization pattern on each chromosome. The method is superior to Giemsa- or fluorescence-based banding methods for chromosome identification because the hybridization patterns are simpler and easier to identify, and unique patterns can be designed at will for each chromosome. Analysis can be performed with a standard fluorescence microscope, and images can be recorded on film with an ordinary 35-mm camera, making the method useful to many investigators. The method can also be applied to any species for which chromosomes and probes can be prepared.     

Discrimination of aneuploidogens from clastogens by C-banding, DNA and area measurements of micronuclei from mouse bone marrow.

Micronuclei (MN) obtained from mouse bone marrow cells, in vivo exposed to 3 typical clastogens (procarbazine, azathioprine, ethyl methanesulfonate) and 3 typical aneuploidogens (vinblastine, tubulazole, colchicine), were examined for C-band, area and DNA content. C-banding allows a clear discrimination between clastogens and aneuploidogens: the clastogens do not exceed 50% C-band-positive MN and the aneuploidogens all 3 produce 65-75% C-band-positive MN. Concerning the DNA content the percentages of MN containing more DNA than an average chromosome (chr) are lower than 12% for the clastogens and 38-60% for the aneuploidogens. As far as the area of the MN is concerned the percentages of MN which have a larger area than chr are lower than 23% for the clastogens and range from 47% to 71% for the aneuploidogens. Additionally 3 other mutagens were studied. Hydroquinone induces 43% C-band-positive MN with DNA content far below the content of chr; considering the area measurements, however, hydroquinone behaves as an aneuploidogen (65% of the MN are larger than chr). Mitomycin C lies between the clastogens and the aneuploidogens for all 3 criteria but 5-azacytidine is comparable to the model aneuploidogens.     

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.