Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50 -80%) are highly mobile (recovery halftime: t 1/2 Ϸ 0.9 s; diffusion coefficient: D Ϸ 0.6 -0.7 m 2 s ؊1 ). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t 1/2 Ϸ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8 -14% of GFP-HP1␣, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1␣ in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle. INTRODUCTIONThe genomic DNA within the eukaryotic nucleus is organized into structurally distinct domains that regulate gene expression and chromosome behavior (Lamond and Earnshaw, 1998). Chromosomes are composed of two types of domains: heterochromatin and euchromatin (Cohen and Lee, 2002;Grewal and Moazed, 2003). Constitutive heterochromatic domains at centromeres and telomeres consist of repetitive DNA and are largely transcriptionally silent. Euchromatin defines the gene-rich and transcriptionally active region of the cell nucleus (Grewal and Elgin, 2002). Heterochromatin mediates many diverse functions in the cell nucleus, including centromere function, gene silencing, regulation of gene expression, and nuclear organization. At centromeres, heterochromatin is required for proper sister chromatid cohesion and mitotic segregation (Bernard et al., 2001;Peters et al., 2001; Nonaka et al., 2002;Hall et al., 2003). Smaller heterochromatin domains are involved in epigenetic regulation of gene expression during development and cellular differentiation (Cavalli, 2002;Grewal and Moazed, 2003). Heterochromatic inactivation of one of the two X chromosomes, giving rise to the Barr body, is essential in dosage compensation in somatic cells of female mammals (Avner and Heard, 2001). The link between heterochromatin and transcriptional silencing has been firmly established by detailed analysis of the phenomenon PEV (position effect variegation), in which a gene is silenced by positioning it abnormally close to heterochromatin (Wallrath and Elgin, 1995).The establishment of heterochromatin requires the physical coupling of histone-modifying activities and structural proteins at specific genomic sites (Richards and Elgin, 2002). The "histone code" hypothesis predicts tha...

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase ox-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase a-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase oe-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase a-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase ft-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase ax-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase aL-primase had been purified. However, the human DNA polymerase a-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.Replication of simian virus 40 (SV40) DNA in infected monkey cells has served as a useful model system for mammalian chromosomal replication (for reviews, see references 1, 10, 19, 34, and 64). The viral genome is complexed with histones as a minichromosome and replicates in the S phase of the cell cycle. Initiation of SV40 DNA synthesis takes place within a short, well-characterized origin region in the viral genome; elongation proceeds bidirectionally from the origin and terminates when the two replication forks meet. Except for a single viral protein, the large tumor (T) antigen, SV40 DNA is replicated by cellular proteins.Biochemical studies of SV40 DNA replication have been facilitated by a cell-free replication assay (38,65,78). Extracts of primate cells, but not of other mammalian cells, were found to support replication of SV40 DNA in vitro, reflecting their viral DNA replication activity in cell culture (39). Fractionation of human cell extracts permitted the isolation of a set of seven cellular proteins that were sufficient to reconstitute viral DNA replication in vitro (35,69,74). Only three of these proteins, DNA polymerase ox-primase, the single-stranded DNA-binding protein RP-A, and topoisomerase I, are sufficient, together with T antigen, to initiate viral DNA replication (9,25,35,41,45,69,75,79 unwinding of the template catalyzed by the DNA helicase activity of T antigen (2,3,8,9,15,16,18,20,54,55,57,63,80). RP-A binds to the parental single strands, topoisomerase I releases torsional stress generated by unwinding, and DNA polymerase oa-primase synthesizes the first primers on both strands. Subsequently, DNA polymerase delta and its accessory proteins assemble at the primer-template junction to form the leading-strand replication complex (69,71,72). DNA polymerase cx-primase and possibly a third DNA polymerase, synthesize the lagging strands (9,52,53,69,75).These events occur in an ordered se...

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