Paul E. Nisson

CCG REPEATS IN cDNAs FROM HUMAN BRAIN

Abstract Expansion mutations of trinucleotide repeats and other units of unstable DNA have been proposed to account for at least some of the genetic susceptibility to a number of neuropsychiatric disorders, including bipolar affective disorder, schizophrenia, autism, and panic disorder. To generate additional candidate genes for these and other disorders, cDNA libraries from human brain were probed at high stringency for clones containing CCG, CGC, GCC, CGG, GCG, and GGC repeats (referred to collectively as CCG repeats). Some 18 cDNAs containing previously unpublished or uncharacterized repeats were characterized for chromosomal locus, repeat length polymorphism, and similarity to genes of known function. The cDNAs were also compared with the 37 human genes with eight or more consecutive CCG triplets in GenBank. The repeats were mapped to a number of loci, including 1p34, 2p11.2, 2q30–32, 3p21, 3p22, 4q35, 6q22, 7qter, 13p13, 17q24, 18p11, 19p13.3, 20q12, 20q13.3, and 22q12. Length polymorphism was detected in 50% of the repeats. The newly cloned cDNAs include a complete transcript of human neurexin-1B, a portion of BCNG-1 (a newly described brain-specific ion channel), a previously unreported polymorphic repeat located in the 5’ UTR region of the guanine nucleotide-binding protein (G-protein) β2 subunit, and a human version of the mouse proline-rich protein 7. This list of cDNAs should expedite the search for expansion mutations associated with diseases of the central nervous system.

Reversible Tumorigenesis Induced by Deficiency of Vasodilator-Stimulated Phosphoprotein

ABSTRACT Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilator-stimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.

Identification of the chromosome 14 origin of a C‐negative marker associated with a 14q32 deletion by chromosome painting

A constitutional chromosome 14 rearrangement was observed in a female with a psychodevelopmental disorder. Karyotype analysis using a variety of chromosome techniques, QFQ, GTG, CBG, Ag‐NOR and DA‐DAPI, showed a deletion of chromosome 14q32.1‐qter region in association with a supernumerary marker chromosome. The marker, resembling a submetacentric, approximately half the size of a G group chromosome is C band and Ag‐NOR negative. The heteromorphism of the satellites showed that the deleted chromosome 14 is paternal in origin. Chromosome painting using an Alu‐PCR probe specific for the human chromosome 14 and fluorescent in situ hybridization (FISH) showed that the marker contains chromosome 14q32 sequences. It is likely that the marker was generated from the deleted chromosome 14 region through a complex rearrangement.

Molecular Cloning of PCR Products

It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high-quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.

Isolation of Exons from Cloned DNA by Exon Trapping

Exon trapping is an RNA polymerase chain reaction (PCR) method to clone expressed sequences or exons directly from mammalian genomic DNA. The basic protocol in this unit describes the method for trapping internal exons from cosmid clones and the second basic protocol describes trapping of 3 terminal exons. An describes 3 terminal exon trapping, which avoids subcloning of target DNA by ligating it to the vector for direct transfection. A describes a rapid cloning procedure using uracil DNA glycosylase.