Heterochromatin and germ line-restricted DNA.
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Biomedical subjects
Publications and source records attributed to W Hennig.
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The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.
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The transcript morphology in the lampbrush chromosome loop pairs ;threads' and ;pseudonucleolus' of the Y chromosome in primary spermatocytes of Drosophila hydei has been investigated by the Miller spreading technique. Each loop pair carries giant transcripts with a secondary structure characteristic of the respective loop. The lengths of the transcribed regions are in the range 500-1500 kb or even larger. The results of our experiments are discussed in the context of loop ultrastructure, molecular structure and loop function. The high degree of secondary structure may be required to assemble specifically loop-specific proteins.
By immunofluorescence techniques and protein blotting experiments we have shown that an antiserum specifically reacts with a Mr 80,000 protein (the "Ps protein") in the lampbrush loop " pseudonucleolus " in spermatocyte nuclei of Drosophila hydei. Comparative studies of X/Y and X/0 testes indicate that the gene encoding the Ps protein is not located on the Y chromosome but on an autosome or the X chromosome. The Ps protein is tissue specific. It is likely to be a rather conserved protein since the antigenic determinant recognized by the antiserum could be detected in the spermatocyte nuclei of a number of other Drosophila species. For those species with prominent Y chromosomal lampbrush loops, it could be shown that the cross-reaction is, as in D. hydei, associated with a specific Y chromosomal loop.
Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.
We microdissected a Y chromosomal lampbrush loop pair from primary spermatocyte nuclei of Drosophila hydei and cloned the DNA directly at the microscale. Four of the 12 recombinant DNA clones recovered display in situ hybridization to mitotic metaphase Y chromosomes, preferentially in the chromosomal region identified as the origin of the lampbrush loop pair. All clones, however, also hybridize to autosomal and X chromosomal loci in polytene chromosomes. Y chromosomal DNA sequences of D. hydei again prove to be members of different families of repeated sequences distributed throughout the genome. These microcloning experiments, which were carried out under very unfavourable experimental conditions (low DNA content of the lampbrush loops in the presence of large amounts of RNA) prove that almost any chromosomal structure detected by light microscopy is directly accessible to molecular cloning experiments by micromethods.
A comparative study of the protein patterns in testes of wild-type and X/O flies of Drosophila hydei revealed quantitative differences in at least three major protein fractions. One protein component of a Mr 155,000 fraction and a protein of Mr 35,000 are completely absent in X/O testes. The amount of protein in a Mr 55,000 fraction is considerably reduced. The tubulins, which are part of this fraction, are also reduced in amount. All three proteins were found as constituents of sperm tails. Studies of Y chromosomal mutants revealed that the presence of at least two of these proteins depends on the activity of loci O, P, and Q of the Y chromosome. However, preliminary evidence indicates an autosomal location of the genes of these sperm proteins. This suggests a regulatory role of Y chromosomal genes in the production of some major sperm proteins.
By recombinant DNA techniques, a Y chromosomal sequence of Drosophila hydei was isolated. This DNA sequence of 8.93-kilobase length is a member of a family of repetitive sequences located in the short arm of the Y chromosome. Tissue-specific transcripts complementary to the cloned sequence were found in testes RNA. In situ hybridization demonstrated that such transcripts are present in the lampbrush loop pair "nooses" in primary spermatocyte nuclei--a loop pair that is associated with the only fertility gene in the short arm of the Y chromosome.
A genetic map of the Y chromosome of Drosophila hydei has been constructed from deletion/complementation experiments, with the aid of male sterile mutants of the Y chromosome. A central conclusion of our experiments is that not more than a single complementation group can be detected in each of the lampbrush loop forming sites. Additional complementation groups, functionally independent of lampbrush loops, reside between these loci. Six complementation groups have been defined by several methods of mapping. An additional ten complementation groups are indicated, but their exact definition requires further investigation. The "synthetic sterility" of mutations in these ten loci contributes to the difficulty in unequivocally establishing their individual boundaries. Mapping problems also arise from the instability of certain mutants.
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Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use 3H- or 125 I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.
Compound bone cement on a PMMA base with an additive of bioactive glass ceramic particles in different portions and different particle sizes are tested in animal experiments. The tissue reactions to extracorporal polymerized specimens and to in situ polymerized specimens are observed. The experiments with an implantation period up to six months demonstrate a tight bonding between the newly formed osseous tissue and the glass ceramic particles at the interface. The inflammatory reactions in the vicinity of the implant are small. It is the objective of the investigations to improve the adherance of the bone cement at the interface to achieve a more durable anchorage of bone cement in the tissue.
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The positions of the nucleolus organizer regions in metaphase chromosomes of Drosophila hydei were detected by in situ hybridization experiments. In agreement with earlier conclusions the nucleolus of the X chromosome was found to originate in a terminal region of the heterochromatic arm. The Y chromosome contains two nucleolus organizers, one in a terminal postion of the long arm, and the other in the short arm. The implications with respect to the evolution of the Y chromosome are discussed.
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