Functional analysis of the human neurofilament light chain gene promoter.

We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression.     

Isolation and characterization of the oligo(dA-dT) clusters and their flanking DNA segments in the rabbit genome

Duplex DNA containing (dA-dT) clusters² can be bound specifically to poly(U)-Sephadex in high-salt solutions by the formation of a (dA-dT)rU triple helix. By decreasing the salt concentration the DNA is fractionated predominantly on the basis of the length of the (dA-dT) cluster it contains, long clusters being bound tighter than short ones. A stepwise elution protocol was used to obtain three fractions containing (dA-dT) clusters of which the average length is approximately 12, 16 and 23 base-pairs, respectively. Each given fraction probably also contains impure (dA-dT) clusters of greater length than these but which have additional bases inserted in the (dA-dT) sequence. When bound to poly(U) these clusters should give triplexes of equivalent stability to those formed with shorter pure (dA-dT) tracts.Measurement of the distribution of rabbit DNA in these poly(U)-Sephadex fractions as a function of DNA size shows that the three (dA-dT) cluster length-classes are interspersed with one another in the rabbit genome. The short, intermediate and long clusters are spaced approximately once every 6000, 12,000 and 18,000 base-pairs throughout at least 80% of the rabbit genome. There is an average of one (dA-dT) cluster every 3300 base-pairs in rabbit DNA.When rabbit DNA is sheared to 400 base-pairs, 50% of the molecules containing a (dA-dT) cluster contain a sequence reiterated approximately 2 × 10⁵-fold in the rabbit genome although only 20% of unfractionated DNA of this size contains such repeated DNA. The remaining 50% of the clusters are linked to DNA sequences occurring one to five times per haploid genome.     

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.     

The construction of cosmid libraries which can be used to transform eukaryotic cells

Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells.     

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.