Biomedical subjects

M A Schäfer

Publications and source records attributed to M A Schäfer.

12 recordsLinked to original sources

PubMed2006

Patterns of microsatellite variation through a transition zone of a chromosomal cline in Drosophila americana.

Chromosomal rearrangements have been considered as important barriers to gene flow and were often used in the delineation of species. The original taxonomic designation of Drosophila americana americana and Drosophila americana texana is based on the presence/absence of a centric fusion between the X- and fourth chromosomes. D. a. americana presents the derived fused state, whereas Drosophila a. texana presents the freely segregating ancestral state. The degree of genetic separation between the two chromosomal forms is still controversial, with different genetic markers yielding contrasting results even when the same populations were analyzed. Using 27 polymorphic microsatellites, we re-evaluated patterns of genetic differentiation between six D. americana populations sampled through a transition zone of both chromosomal forms in the central United States. Our results clearly reject a scenario of two differentiated species forming a hybrid zone in a region of parapatry and indicate that gene flow minimizes genome-wide differentiation associated with the two chromosomal arrangements.

Animals↗

PubMed2002

The essential Drosophila melanogaster gene wds (will die slowly) codes for a WD-repeat protein with seven repeats.

We have isolated and characterized the will die slowly (wds) gene of Drosophila melanogaster, formerly known as l(1)zw8 or l(1)3Ad. The gene codes for a 2.0-kb RNA that is transcribed at all stages of development. The RNA has been localized by in situ hybridization to imaginal discs, larval brain, to nurse cells in the ovary, and to spermatogonia and spermatocytes in the testis. The putative translation product contains seven WD-repeats and is, therefore, a new member of the family of WD-proteins. Clear homologues of the Drosophila WDS protein exist in three other fully sequenced higher eukaryotes - human, Caenorhabditis elegans and Arabidopsis. A genomic fragment containing the wds transcription unit is able to rescue two different lethal wds alleles, thus proving that we have indeed isolated the wds gene.

Amino Acid Sequence↗

PubMed2001

Geographical patterns of genetic subdivision in the cellar spider Pholcus phalangioides (Araneae).

Geographical patterns of gene flow and drift were analysed in the commensal cellar spider Pholcus phalangioides to get insight into the causes affecting genetic variation in this species strictly associated with man. Our sampling consisted of 23 subpopulations collected over five urban regions in central Europe (distances ranged from 920 km to sites within the same building complex). Five variable allozyme loci showed significant interpopulation subdivision (theta=0.146) and isolation by distance over the area studied. On a regional scale (up to 70 km) significant differentiation was found, but the genetic pattern did not correlate with distance. Moreover, significant two-locus disequilibria were detected and a recent reduction in the effective population size was indicated within six sites. These results suggest that in P. phalangioides a high potential of dispersal and strong effects of drift within small, demographically unstable mating units seem to cause significant, but unpredictable genetic differentiation patterns at lower geographical scales. Our study documents strong effects of drift in a strictly commensal species outside the murine rodents.

Animals↗

PubMed2000

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Amino Acid Sequence↗

PubMed2000

Alterations in titer and distribution of high mobility group proteins during embryonic development of Drosophila melanogaster.

High mobility group proteins are thought to have an architectural function in chromatin. Here we describe changes in titers, extent of phosphorylation, and cellular distribution of the three abundant HMG proteins during embryonic development of Drosophila. The titers of the HMG proteins HMGD, HMGZ, and D1 are highest in ovaries and at the beginning of embryonic development. They decrease continuously until cellularization of the embryo. Relative to the histone H1 titer, the levels of HMGD and D1 remain almost constant during gastrulation and organogenesis, whereas the titer of HMGZ increases during late organogenesis. Up to gastrulation, the development is accompanied by dephosphorylation of D1. In contrast, HMGD and HMGZ appear to be constitutively phosphorylated. As the high extent of phosphorylation of D1 is also characteristic in ovaries, it is likely that the posttranslational modifications of this protein observed in early embryonic stages are of maternal origin. Using site specific antibodies against helices I and III of HMGD and HMGZ and against the AT-hook motif of D1, protein-specific staining patterns have been observed during embryonic development. Despite high levels of HMG proteins at the beginning of embryonic development, we were unable to detect any of these proteins in nuclei of stage 2 embryos. The accumulation of the HMG proteins correlates with the onset of transcription in stage 3. Our results argue against a proposal of a shared role of HMGD and histone H1 in Drosophila chromatin.

Animals↗

PubMed1998

Protection patterns of tRNAs do not change during ribosomal translocation.

The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.

Binding Sites↗

PubMed1998

The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster.

Oogenesis in Drosophila is a useful model for studying cell differentiation. We have analyzed the role of the egh gene in these processes with the aid of a newly isolated viable but female sterile allele. This mutation results in diverse variable defects in oogenesis. The most frequent defect being follicles that have either more or less than the normal number of 16 germ cells. This is caused by erroneous splitting and/or fusion of correct clusters of 16 cystocytes. The entire follicle has a rather flexible structure in this allele, most obvious by a highly variable position of the oocyte within the follicle. Moreover, a second oocyte can also develop in egh clusters. This is exclusively observed in aberrant follicles that are generated by the aforementioned splitting/fusion process. Surprisingly, even a germ cell which is distinct from the two pro-oocytes can differentiate into an oocyte under these circumstances. Hence, determination of the oocyte is definitely not fixed when germ cell clusters are enveloped by prefollicular cells, and interactions between follicle cells and germ cells must play an important role in oocyte specification. Molecular analysis proves that the oocyte-specific transcript of the egh gene is drastically reduced in this viable allele.

Animals↗

PubMed1998

Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila.

The aly, can, mia and sa genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In contrast can, mia and sa are required for accumulation of twine protein but not twine transcript. We propose that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. We also propose that control of translation or protein stability regulates entry into the first meiotic division. We suggest that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of twine protein, thus coordinating meiotic division with onset of spermatid differentiation.

Animals↗

PubMed1997

Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element).

Animals↗

PubMed1996

Mutational analysis of two highly conserved UGG sequences of 23 S rRNA from Escherichia coli.

The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3'-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (Psi2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.

Base Sequence↗

PubMed1996

Conserved nucleotides of 23 S rRNA located at the ribosomal peptidyltransferase center.

Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schäfer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849-32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.

Base Sequence↗

PubMedDate not supplied

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Amino Acid Sequence↗