Janine M. LeBlanc-Straceski

A second-generation YAC contig map of human chromosome 12.

Breakthrough: The Quest to Isolate the Gene for Hereditary Breast Cancer.By Kevin Davies and Michael White. Mac-millan: 1995. Pp. 370. £16.99. To be published in the United States by Wiley.

Developmental expression of Xenopus myosin 1d and identification of a myo1d tail homology that overlaps TH1

Xenopus laevis myosin 1d (XlMyo1d) is a member of the myosin I class, subclass 4. Members of this class are single headed, bind calmodulin light chains and have lipid binding domains in their tails. The rat myo1d homologue has been implicated in endosome vesicle recycling in epithelial cells. Mutations in the Drosophila myosin 1d homologue cause situs inversus in the abdomen. The XlMyo1d cDNA has been cloned and the derived amino acid sequence is 80% identical to the rat and human homologues. Sequence comparison revealed a novel isoform‐specific tail homology embedded in the Tail Homology 1 (TH1) domain characteristic of myosin I isoforms. Western blot analysis using a polyclonal antibody raised against an isoform‐specific peptide showed that the protein is present in eggs and levels increase at early neurula through tadpole stages. Whole mount in situ hybridization using a probe containing the 5′UTR (untranslated region) showed that XlMyo1d mRNA is expressed in neural tube, pre‐somitic mesoderm, somites and all three segments of cranial neural crest cells during their migration. Sections of the in situ hybridizations revealed that during somitogenesis, XlMyo1d mRNA was localized to a stripe overlapping the nuclear region of somites during early tadpole stages.

The lift pool method for isolation of cDNA clones from lambda phage libraries

A PCR based strategy was developed to screen a Xenopus oocyte λgt10 cDNA library. The PCR-based lift pool (LP) method follows the same two tiered strategy as conventional screening of phage libraries by filter hybridization. Two rounds of plating, one at high density to detect the clone, and one at low density to purify the clone to homogeneity, are performed. In the first round, lysates from high density plates, termed plate pools (PP), serve as template for PCR. In the second round, phage particles adhering to plaque lifts of low density plates are washed off nitrocellulose filters to create LPs, which are used as template for PCR. The integrity of the plaques on the low-density plates is preserved. Once a positive LP has been identified, plaques on the corresponding plate are screened individually by PCR. Using isoform specific primer pairs for Xenopus myosin 7a and myosin 1d, two lambda clones were isolated. Subsequent DNA sequence analysis confirmed their identities as myosin isoforms (GenBank accession numbers: DQ100353 and AF540952). This method offers a time saving, cost-effective alternative to other hierarchical pooling strategies for the repeated screening by PCR of an arrayed lambda phage library.