Molecular evolution of the ZFY and ZNF6 gene families

Members of the ZFY and ZNF6 gene families have been cloned from species representing different taxa and different modes of sex determination. Comparisons of these genes show the ZFY-like and ZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequence analyzed by neighbor-joining indicated that both gene families are monophyletic with a high bootstrap value. Pairing of sequences from males and females of nonmammalian species showed there to be no significant difference between male and female sequences from a single species, consistent with autosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-like sequences suggested that Zfy genes have undergone a period of rapid evolutionary change not seen in human ZFY.      

Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast

A checkpoint responding to DNA damage in G2 results in a delay in the onset of mitosis through inhibition of p34^cdc2 kinase activity via maintenance of inhibitory tyrosine phosphorylation. Genetic analyses of this checkpoint in fission yeast have identified single alleles of several genes, suggesting these screens are not yet saturating, and hence further genes await identification. To fully understand the complexity of this checkpoint it will be necessary to define all the genes involved. To this end we screened for new mutants defective in the ability to delay mitosis in the presence of DNA-damaging agents. Twenty-four mutants were isolated that were defective in UV-C and MMS-induced checkpoint delay. Amongst these mutants was an allele of cut5 that was also defective in the checkpoint responses. We show here, contrary to previous reports, that the UV-C induced checkpoint response is defective in cut5 mutants. Therefore, like all other checkpoint mutants, cut5 is required for G2 checkpoint arrest following DNA damage, regardless of the nature of the lesions involved. Received: 24 July 1998 / Accepted: 14 September 1998     

An immunoglobulin-like domain determines the specificity of neurotrophin receptors.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.     

Phosphorylation in vivo of four basic proteins of rat brain myelin

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H₃³²PO₄. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [³²P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.