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.

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.

Sam68 is a multimeric 68-kDa RNA-binding nuclear protein of unknown function that interacts with, and is tyrosine-phosphorylated by, the oncogenic protein Src during mitosis. Random homozygous knock-out (RHKO) is a retroviral-based antisense RNA strategy that can identify chromosomal genes whose functional disablement leads to reversible tumorigenic capabilities. Here we report that RHKO-induced Sam68 deficiency results in neoplastic transformation of murine NIH3T3 fibroblasts. Whereas simple haploinsufficiency of Sam68 produced by insertion mutagenesis in a single chromosomal allele did not detectably affect cell growth, reduction of Sam68 protein to <25% of the wild type level was associated with anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice. These properties were reversed by cessation of RHKO inactivation. Our findings, which indicate that the Sam68 protein level can prominently affect cell proliferation, implicate Sam68 function in tumorigenesis. Consistent with these results is evidence that cells undergoing mitosis show a dramatic reduction in the level of Sam68 protein.

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.

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.

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase zeta, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of approximately 340 residues, 39% identical in a carboxyl-terminal region of approximately 850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol zeta type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol zeta among all polymerases in the GenBank database, and is different from the human alpha, delta, and epsilon enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.

The t(8;21) translocation is associated with a high percentage of acute myelogenous leukemia (AML) cases of type 2 FAB. This cytogenetic landmark has been instrumental in the positional cloning of the AML1 gene which encodes a transcription factor and spans the translocation region. Using 3{prime} RACE and exon trapping, multiple AML1 transcripts have been observed which are generated by alternative splicing 3{prime} to exon 5. Although several transcripts from the AML1 gene have been cloned, these account for only a fraction of those predicted by Northern blotting. We therefore have subjected a 240 kb YAC (C4C10) that contains the entire AML1 gene to internal and 3{prime}-terminal exon trapping in an attempt to fully characterize the transcript repetoire from AML1. Exon trapping has been shown previously to capture exonic sequence by selecting splicing signals and has been applied primarily on cosmids. We report here the development of protocols for the efficient capture of internal and 3{prime}-terminal exons from the AML1 gene directly from YAC DNA.

The t(8;21) translocation is associated with a high percentage of acute myelogenous leukemia (AML) cases of type 2 FAB. This cytogenetic landmark has been instrumental in the positional cloning of the AML1 gene which encodes a transcription factor and spans the translocation region. Using 3{prime} RACE and exon trapping, multiple AML1 transcripts have been observed which are generated by alternative splicing 3{prime} to exon 5. Although several transcripts from the AML1 gene have been cloned, these account for only a fraction of those predicted by Northern blotting. We therefore have subjected a 240 kb YAC (C4C10) that contains the entire AML1 gene to internal and 3{prime}-terminal exon trapping in an attempt to fully characterize the transcript repetoire from AML1. Exon trapping has been shown previously to capture exonic sequence by selecting splicing signals and has been applied primarily on cosmids. We report here the development of protocols for the efficient capture of internal and 3{prime}-terminal exons from the AML1 gene directly from YAC DNA.

The t(3;21)(q26;q22) is associated with chronic myelogenous leukemia in blast crisis (CML-BC), leukemia evolving from (therapy-related) myelodysplasia, and with leukemia following other hematopoietic proliferative diseases. Molecular cytogenetic analysis and cloning of a few t(3;21) cases indicate that the breakpoints are quite heterogeneous even within a specific clinical phenotype. Interestingly some of the (3;21) breakpoints involve the AML1 gene previously found rearranged in the t(8;21) associated with acute myelogenous leukemia. AML1 is related to the Drosophila gene runt and is the human counterpart of the gene for the alpha subunit of the nuclear polyoma enhancer binding protein (PEBP2) also known as the core binding factor (CBF). In the t(3;21) AML1 was found rearranged with EAP, a gene on chromosome 3 encoding a small ribosomal protein, as well as with EV11, another gene on chromosome 3. Here we report our study of six cases of t(3;21). By using fluorescence in situ hybridization (FISH) analysis and AML1 probes we could conclude that at least in two CML-BC cases the breakpoint occurred in the AML1 intron that is disrupted by the t(8;21). An AML1/EAP fusion transcript, different from the one described in a therapy-related myelodysplasia, was detected in both CML-BC cases. This transcript is expected to result in a predicted protein containing the AML1 nuclear binding domain with an attached stretch of 17 amino acids unrelated to the EAP small ribosomal protein. In the other t(3;21) patients we could not detect an AML1/EAP transcript or an AML1/EV11 transcript. This result suggests heterogeneity of the t(3;21) at the molecular level. The AML1 chimeric transcripts identified so far, both in the t(3;21) and in the t(8;21), diverge from the normal transcripts either after exon 5 or exon 6. Here we show that in normal AML1 transcripts different splicing events are seen to occur after AML1 exon 5 as well as exon 6.

P E Nisson, A Ally, and P C Watkins Life Technologies, Inc., Gaithersburg, Maryland 20884-9980, USA.

A method for efficient nonradioactive labeling of DNA with biotin using random primer extension has been developed. Under the conditions described, a significant amount of DNA synthesis occurs during incorporation of the nonradioactive label, resulting in amplification of the original template DNA. The effect of primer size, substrate concentration, enzyme concentration, and ratio of biotinylated nucleotide to normal nucleotide on the amount of DNA synthesis was determined. Amplifications of 10- to > 300-fold were attained, depending on the starting template concentration. Template may be varied from 1 to 500 ng per reaction. The size of the resulting biotinylated probes is 100-1000 nucleotides with a significant proportion in the 100-300 nucleotide range. The biotinylated probes were used to detect single-copy genes on Southern blot hybridizations and to identify specific loci in metaphase chromosome spreads by in situ hybridization followed by fluorescent detection with streptavidin-fluorescein isothiocyanate. Random primer amplification and labeling provides a convenient method for preparation of biotinylated probes from small amounts of template DNA.