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A number of examples of putative eukaryote-to-prokaryote horizontal gene transfer (HGT) have been proposed in the past using phylogenetic analysis in support of these claims but none have attempted to map these gene transfers to the presence of genomic islands (GIs) in the host. Two of these cases have been examined in detail, including an ATP sulfurylase (ATPS) gene and a class I fructose bisphosphate aldolase (FBA I) gene that were putatively transferred to cyanobacteria of the genus Prochlorococcus from either green or red algae, respectively. Unlike previous investigations of HGT, parametric methods were initially used to detect genomic islands, then more traditional phylogenomic and phylogenetic methods were used to confirm or deny the HGT status of these genes. The combination of these three methods of analysis- detection of GIs, the determination of genomic neighborhoods, as well as traditional phylogeny, lends strong support to the claim that trans-domain HGT has occurred in only one of these cases and further suggests a new insight into the method of transmission of FBA I, namely that cyanophage-mediated transfer may have been responsible for the HGT event in question. The described methods were then applied to a range of prochlorococcal genomes in order to characterize a candidate for eukaryote-to-prokaryote HGT that had not been previously studied by others. Application of the same methodology used to confirm or deny HGT for ATPS and FBA I identified a ⊗12 fatty acid desaturase (FAD) gene that was likely transferred to Prochlorococcus from either green or red algae.

Recently, the existence of a 'histone code' has been proposed to explain the link between the covalent chemical modification of histone proteins and the epigenetic regulation of gene activity. Although the role of the four 'core' histones has been extensively studied, little is known about the involvement of the linker histone, histone H1 and its variants, in this code. For many years, few sites of chemical modification had been mapped in linker histones, but this has changed recently with the use of functional proteomic techniques, principally mass spectrometry, to characterize these modifications. The functionality of many of these sites, however, remains to be determined.

We find that reconstituted nucleosome cores containing specific DNA sequences dissociate on dilution. This disruption of histone-DNA contacts leading to the release of free DNA is facilitated by the presence of the core histone tails, MgCl2 (5 mM), KCl (60 mM), and temperatures above 0°C. Under reaction conditions that are commonly used to assess trans-acting factor access to nucleosomal DNA, histone-DNA contacts are on the threshold of instability. We demonstrate how dilution of reconstituted nucleosomes containing a TATA box can facilitate TBP access to DNA.

We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.

The process of development can be viewed as a series of linker histone replacements which take place throughout spermatogenesis and oogenesis, as well as following fertilization or somatic nuclear transfer (SNT). Although few of the histone H1 variants in question have been shown to be essential for viability, the timing of their appearance as well as the affinity with which they are able to bind to chromatin seem to be important factors in their developmental role. A looser binding of linker histones to chromatin seems to correlate with the meiotic phases of gametogenesis and the establishment of a totipotent, as well as the maintenance of a pluripotent, state in early embryos, while tighter binding of linker histones to chromatin appears to be associated with the mitotic phases, as well as the increased levels of condensation that are required for the packaging of DNA into sperm. This latter process also involves the binding of certain basic non-histone proteins to DNA. While all proteins involved in chromatin compaction during development are highly basic in nature, in general they can be seen to change from lysine-rich variants to arginine-rich ones, and back again. The fact that linker histone transitions are conserved across diverse metazoan species speaks of their importance in packaging DNA in a variety of ways during this crucial period.

We have found direct DNA repeats 21-47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids > or = 40 kb in length.

Expansion and methylation of CGG repeat sequences is associated with Fragile X syndrome in humans. We have examined the consequences of CGG repeat expansion and methylation for nucleosome assembly and positioning on the Fragile X Mental Retardation gene 1 (FMR1) gene. Short unmethylated CGG repeats are not particularly favored in terms of affinity for the histone octamer or for positioning of the reconstituted nucleosome. However, upon methylation their affinity for the histone octamer increases and a highly positioned nucleosome assembles with the repeat sequences found adjacent to the nucleosomal dyad. Expansion of these CGG repeats abolishes the preferential nucleosome assembly due to methylation. Thus, the expansion and methylation of these triplet repeats can alter the functional organization of chromatin, which may contribute to alterations in the expression of the FMR1 gene and the disease phenotype.

Expansion of CTG repeat sequences is associated with several human genetic diseases. We have examined the consequences of CTG repeat expansion for nucleosome assembly and positioning. Short CTG repeats are found within the most favored DNA sequences yet defined for nucleosome assembly. We find that as few as six CTG repeats will facilitate nucleosome assembly to a similar extent as the 50 or more repeats found in disease genes. Thus an increase in nucleosome stability on expansion of existing triplet repeats is unlikely to explain the acquisition of the disease phenotype. However, the CTG repeat sequence is efficiently wrapped around the histone octamer, preferring to associate with histones at the nucleosomal dyad. Thus short segments CTG repeat sequence will facilitate the assembly of a stable positioned nucleosome which might contribute to the expansion phenomenon and the functional organization of chromatin.

We establish that the TATA binding protein (TBP) in the presence of TFIIA recognizes the TATA box in nucleosomal DNA dependent on the dissociation of the amino-terminal tails of the core histones from the nucleosome and the position of the TATA box within the nucleosome. We examine TBP/TFIIA access to the TATA box with this sequence placed in four distinct rotational frames with reference to the histone surface and at three distinct translational positions at the edge, side and dyad axis of the nucleosome. Under our experimental conditions, we find that the preferential translational position at which TBP/TFIIA can bind the TATA box is within linker DNA at the edge of the nucleosome and that binding is facilitated if contacts made by the amino-terminal tails of the histones with nucleosomal DNA are eliminated. TBP/TFIIA binding to DNA at the edge of the nucleosome occurs with the TATA box in all four rotational positions. This is indicative of TBP/TFIIA association directing the dissociation of the TATA box from the surface of the histone octamer.

For over a century, the origin of eukaryotes has been a topic of intense debate among scientists. Although it has become widely accepted that organelles such as the mitochondria and chloroplasts arose via endosymbiosis, the origin of the eukaryotic nucleus remains enigmatic. Numerous models for the origin of the nucleus have been proposed over the years, many of which use endosymbiosis to explain its existence. Proposals of microbes whose ancestors may have served as either a host or a guest in various endosymbiotic scenarios abound, none of which have been able to sufficiently incorporate the cell biological as well as phylogenetic data which links these organisms to the nucleus. While it is generally agreed that eukaryotic nuclei share more features in common with archaea rather than with bacteria, different studies have identified either one or the other of the two major groups of archaea as potential ancestors, leading to somewhat of a stalemate. This paper seeks to resolve this impasse by presenting evidence that not just one, but a pair of archaea might have served as host to the bacterial ancestor of the mitochondria. This pair may have consisted of ancestors of both Ignicoccus hospitalis as well as its ectosymbiont/ectoparasite ‘Nanoarchaeum equitans’.