Biomedical subjects
Publications and source records attributed to S M Gasser.
The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.
We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentrations of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented.
Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix.
Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions. We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold. Here, we extend these observations to the fission yeast Schizosaccharomyces pombe. In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species. In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species. Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both.
We have used monospecific antibodies against three ribonuclease H enzymes of Saccharomyces cerevisiae to investigate their intracellular localization. Fractionation experiments, as well as immunocytochemical staining, revealed a predominantly cytoplasmic localization of the RNase H proteins of 42,000 and 70,000 Mr, whereas that of 55,000 Mr showed equal distribution between nuclei and cytoplasm. The nuclear moiety of ribonuclease H(70) was found to be a part of the yeast nuclear scaffold, as investigated by immunoblotting and antibody inhibition experiments. The 42,000 and 55,000 Mr enzymes, on the other hand, are not scaffold-associated. We conclude that RNase H(70) is part of the nuclear substructure of yeast that was previously found to maintain specific interactions with yeast chromosomal origins of replication (ARS elements).
DNA fragments containing the silencers that flank the mating type genes at HML alpha are shown to bind specifically to the nuclear scaffold of yeast. The scaffold proteins are solubilized with urea and then renatured to form a soluble extract which allows reconstitution of sequence-specific DNA loops. At the silent mating type locus HML alpha, loops are formed by either silencer-silencer (E-I) interaction or silencer-promoter interactions (E-P and I-P). The nuclear protein RAP-1 fractionates efficiently with the nuclear scaffold, and binds to the E, I, and promoter regions. Affinity purification of RAP-1 and oligonucleotide competition show that RAP-1 is necessary for reconstitution of loops in vitro. These results are consistent with a model in which silencers define a chromatin loop within which occur modifications that maintain the promoter in an inactive state.
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We describe here for the first time the isolation of a yeast nuclear scaffold that maintains specific interactions with yeast genomic DNA sequences. The scaffold-DNA interaction is reversible and saturable, and some binding sites are conserved between yeast and Drosophila KC cells. Second, we find that the specific sequences bound to the yeast nuclear scaffold are the putative origins of replication (ARS elements) and a chromosomal centromere, CENIII. The scaffold association has been closely mapped at the ARS1 locus, and appears to include the 11 bp ARS consensus, but not the ABF-1 binding site. Competition studies show that ARS1 does not compete for CENIII binding, allowing us to distinguish two classes of scaffold attachment sites by functional and structural criteria.
We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.
We have been able to map specific DNA fragments at the bases of chromatin loops with the help of a novel extraction procedure by using lithium-3',5'-diiodosalicylate. One such scaffold-attached region (SAR) is found in the non-transcribed spacer in each repeat of the histone gene cluster, on a 657 base pair (b.p.) restriction fragment. Exonuclease III digestion has localized two protein-binding domains on the SAR of the histone cluster. Each covers approximately 200 b.p. and they are separated by a nuclease-accessible region of about 100 b.p. These domains are rich in sequences closely related to the topoisomerase II cleavage consensus. We have studied the scaffold association of three developmentally regulated genes of Drosophila melanogaster: alcohol dehydrogenase (Adh), the homoeotic gene fushi tarazu (ftz) and Sgs-4, a gene encoding one of the glue proteins secreted by third-instar larvae. We find regions attached to the nuclear scaffold (SARS) both 5' and 3' of all three genes, defining small domains ranging from 4.5 to 13 kilobases. In the case of Adh, a gene with two promoters, we find two upstream and two downstream SARS. Those 5' of the gene co-map with regulatory regions for the adult and the larval transcripts, respectively. For Sgs-4, the 5' SAR covers 866 b.p. immediately upstream of the transcript, and encompasses the 200 b.p. regulatory region defined by two deletion mutants that produce little or no Sgs-4 protein. In ftz the 5' SAR is found 4.8 kilobases upstream of the start of transcription within a 2.5 kilobase element required for a high level of ftz expression in the early embryo. Sequence analysis of five upstream SARS reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II. In addition, they contain multiple copies of two sequence motifs: a specific 10 b.p. A-rich sequence, and another 10 b.p. T-rich stretch. In conclusion, the intimate association of the SAR with the upstream/enhancer elements, the presence of clustered sequences highly homologous to the topoisomerase II cleavage consensus, and the localization of topoisomerase II in the scaffold, suggest a structure-function relation between chromosome organization and gene expression.
We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gei patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.
We find DNA fragments attached to the nuclear scaffold (SARs) both 5' and 3' of three Drosophila genes, defining looped domains ranging from 4.5 to 13 kb. For the two-promoter-containing gene Adh (alcohol dehydrogenase), we find two upstream and two downstream SARs. For Sgs-4, the 5' SAR covers 866 bp immediately upstream of the transcript, and in the case of fushi tarazu, the 5' SAR is found on a small fragment 4.8 kb upstream of the start of transcription. These four upstream scaffold-attached fragments comap with enhancer-like regulatory sequences. Sequence analysis of five upstream SARs reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II, several copies of a specific 10 bp A-rich sequence (AATAAATCAAA), and another 10 bp T-rich stretch.
SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.
Previous experiments have identified a 657-bp restriction fragment in the non-transcribed region of the Drosophila histone gene cluster that is specifically associated with the histone-depleted nuclear scaffold. The remaining fragments of the 5-kb histone repeat were shown to be readily released from the scaffold; hence it was proposed that the tandemly repeated cluster of histone genes forms a series of 5-kb loops restrained by a nuclear substructure at the sites of attachment. Here we show that the attachment fragment is tightly associated with protease-sensitive material, whereas the solubilized fragments are relatively protein-free. Exonuclease III digestion has been used to map the location of protein complexes on the attachment fragment. We have defined two regions of approximately 200 bp whose borders provide kinetic barriers to exonuclease III degradation. They are separated by a nucleaseaccessible region of approximately 100 bp. The protected regions are sufficient to mediate association of the fragment with the histonedepleted nuclei. Sequence analysis reveals an enrichment for sequences closely related to the topoisomerase II cleavage consensus in these two domains.
The mitochondrial ubiquinol--cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm. We have studied the synthesis of five of the nuclear-encoded subunits both in vivo and in vitro and show that of these the 44-kDa, 40-kDa and 17-kDa subunits are synthesized with cleavable extensions, while the 14-kDa and 11-kDa proteins are synthesized without detectable extra sequences. The sizes of the pre-sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5-kDa for the 44-kDa and 40-kDa subunits to 9-kDa for the 17-kDa subunit. The existence in vivo of precursor forms to the 44-kDa, 40-kDa and 17-kDa subunits implies that import is at least partially a post-translational process. The precursor of the 44-kDa subunit can be processed post-translationally in vitro by isolated mitochondria. The messenger RNAs for subunits of the complex have been studied. Those coding for the 44-kDa, 40-kDa, 14-kDa and 11-kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.
The yeast mitochondrial outer membrane contains a major 29-kilodalton protein which is encoded in the nucleus. When this polypeptide is synthesized in vitro and then incubated with isolated yeast mitochondria, it binds to the organelles and becomes resistant to externally added trypsin. Post-translational insertion of the 29-kilodalton protein in vitro is not accompanied by NH2-terminal processing, appears to require neither ATP nor a transmembrane electrochemical potential, and is also observed with purified outer membranes, but not with yeast microsomes. In vitro insertion of the 29-kilodalton polypeptide is thus in several respects different from that of polypeptides destined for the internal compartments of the mitochondrion.
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