Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts.

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

Characterisation of transcriptionally active and inactive chromatin domains in neurons.

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These data indicate that neuronal, perinucleolar heterochromatin consists of several classes of inactive DNA, that are linked to a fraction of the inactive rDNA repeats. These distinct chromatin domains may serve to regulate RNA transcription and processing efficiently and to protect rDNA repeats against unwanted silencing and/or homologous recombination events.

The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains.

Murine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Krüppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abundant in the developing central nervous system, and in the adult mouse expression is restricted largely to testis and brain. Here we show that at the protein level ZFP-37 is detected readily in neurons of the adult central nervous system but hardly in testis. In brain ZFP-37 is associated with nucleoli and appears to contact heterochromatin. Mouse and human ZFP-37 have a basic histone H1-like linker domain, located between KRAB and zinc finger regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a structural protein of the neuronal nucleus which plays a role in the maintenance of specialized chromatin domains.

Amphotropic retroviruses with a hybrid long terminal repeat as a tool for gene therapy of cystic fibrosis.

We have made two retroviral vectors encoding the bacterial beta-galactosidase (lacZ) as a marker gene and a long terminal repeat (LTR) containing an enhancer of the polyoma F101 virus [symbol: see text]. One vector, [symbol: see text], can be used as a test vector in grafting, lineage analysis and gene therapy studies. The other, [symbol: see text] carries an additional unique cloning site in which a gene of interest can be cloned. Titration experiments showed that in human epithelial cell lines, [symbol: see text] produces a transcriptionally active integration more often than the commonly used BAG vector with the wild type LTR. Human epithelial cells in primary culture could be successfully infected. Our data suggest that gene therapy protocols requiring infection in situ, such as in the case of cystic fibrosis, will be hampered by the relatively low local titres that can be achieved at present.

Alignment and focusing unit for dual-laser excitation in the fluorescence-activated cell sorter.

The two laser beams in a dual-laser fluorescence-activated cell sorter FACS-II can be aligned and focused independently on the sample stream with an additional unit, which can be fitted easily on the optical bench of the FACS. The unit consists of two spherical lenses, which have been mounted in separate holders and can be moved in three directions by way of micrometer gauges. The lenses, which have different focal lengths, have been cut off on one side so each laser beam only passes one lens. The setup has been tested using the flow analysis of a suspension of double-stained chicken red blood cells. The histograms of both fluorescence signals showed normal distributions with a coefficient of variation of approximately 6%. After willful interference with the adjustments, the laser beams could be readily readjusted within five minutes.