A Game of Thrones at Human Centromeres I. Multifarious structure necessitates a new molecular/evolutionary model

Human centromeres form over arrays of tandemly repeated DNA that are exceptionally complex (repeats of repeats) and long (spanning up to 8 Mbp). They also have an exceptionally rapid rate of evolution. The generally accepted model for the expansion/contraction, homogenization and evolution of human centromeric repeat arrays is a generic model for the evolution of satellite DNA that is based on unequal crossing over between sister chromatids. This selectively neutral model predicts that the sequences of centromeric repeat units will be effectively random and lack functional constraint. Here I used shotgun PacBio SMRT reads from a homozygous human fetal genome (female) to determine and compare the consensus sequences (and levels of intra-array variation) for the active centromeric repeats of all the chromosomes. To include the Y chromosome using the same technology, I used the same type of reads from a diploid male. I found many different forms and levels of conserved structure that are not predicted by –and sometimes contradictory to– the unequal crossing over model. Much of this structure is based on spatial organization of three types of ~170 bp monomeric repeat units that are predicted to influence centromere strength (i.e., the level of outer kinetochore proteins): one with a protein-binding sequence at its 5’ end (a 17 bp b-box that binds CENP-B), a second that is identical to the first except that the b-box is mutated so that it no longer binds CENP-B, and a third lacking a b-box but containing a 19 bp conserved “n-box” sequence near its 5’ end. The frequency and organization of these monomer types change markedly as the number of monomers per repeat unit increases, and also differs between inactive and active arrays. Active arrays are also much longer than flanking, inactive arrays, and far longer than required for cellular functioning. The diverse forms of structure motivate a new hypothesis for the lifecycle of human centromeric sequences. These multifarious levels of structures, and other lines of evidence, collectively indicate that a new model is needed to explain the form, function, expansion/contraction, homogenization and rapid evolution of centromeric sequences.

Controlled Release of Thrombin Using Aptamer-Based Nanodevices

Aptamers are DNA or RNA single strands that have been selected from random pools based on their ability to bind ligands. Like antibodies, aptamers are highly specific to their targets, and thus have many potential uses in biomedicine and biotechnology. We report here on the construction of a protein-binding molecular device based on a DNA aptamer, which can be instructed to hold or release the human blood-clotting factor, α-thrombin, depending on an operator DNA sequence addressing it. In the operation of this DNA nanodevice, the thrombin-binding DNA aptamer is switched between a binding and a non-binding form. This is achieved by sequentially hybridizing and removing a DNA single strand to the protein binding region of the aptamer. This principle of operation is limited as the switching sequence is determined by the protein-binding sequence. To overcome this limitation we introduce a DNA signal translation device that allows the operation of aptamers with arbitrary sequences. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences.

Isolation of the LEMMI9 Gene and Promoter Analysis During a Compatible Plant-Nematode Interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-β glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

Molecular organization of the interferon γ-binding domain in heparan sulphate

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulphated or N-acetylated. This led us to take a ‘footprinting’ approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.

Identification and characterization of a novel enhancer for the rat neu promoter.

We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.

Identification and characterization of a novel enhancer for the rat neu promoter

We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.