Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1− and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT− cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT− products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1− cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1− cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1− cells are repaired by illegitimate recombination.

Manipulating the mammalian genome by homologous recombination

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies—chimeric nucleases and triplex-forming oligonucleotides—for stimulating recombination in cells.

Diary : manuscript, 1666-1679, 1723, and 1726

Diary concerning chiefly religious matters, mostly Puritanical confessions of Tompson's piety not living up to the expectation of the Lord. There is also mention of the many afflictions God is "pleased" to bestow upon Tompson's wife.

Laboratory investigation of residual liquid organics from spills, leaks, and the disposal of hazardous wastes in groundwater /

Mode of access: Internet.

Sunday reading for christian families and individuals : containing short commentaries, scripture histories, geography ... sacred poetry /

Includes bibliographical references.

High daily rate trickling filter performance : under the direction of the Board of State Health Commissioners, Upper Mississippi River Basin Sanitaton Agreement /

Includes bibliographical references.

Apostolic ministry : sermons and addresses /

Mode of access: Internet.

Strength out of weakness : or A glorious manifestation of the further progress of the gospel among the Indians in New England /

Includes letters from John Eliot, John Wilson, William Leverich, Anthony Bessey, Thomas Mayhew, John Endecott, William French and Thomas Allen. The Epistle dedicatory is signed by William Steele. "To the reader" signed: William Gouge [and seventeen others].

The life of David Lloyd George, with a short history of the Welsh people,

Mode of access: Internet.

La guerra y la democracia,

Mode of access: Internet.

Twelve years' military adventure in three quarters of the globe; or, Memoirs of an officer who served in the armies of His Majesty and of the East India Company, between the years 1802 and 1814, in which are contained the campaigns of the Duke of Wellington in India, and his last in Spain and the south of France.

Mode of access: Internet.

Contemporary France,

Vols. 2-4 have imprint: London, A Constable & co. ltd.

A short inquiry into the policy, humanity and past effects of the poor laws : and into the principles upon which any measures for their improvement should be conducted : in which are included a few considerations on the questions of political oeconomy, most intimately connected with the subject : particularly on the supply of food in England /

Mode of access: Internet.

Merry songs and ballads, prior to the year 1800 /

Head and tail pieces.

Tratado de la fiebre amarilla que desde últimos de agosto hasta principios de octubre del año pasado ha reynado en la banda de San Juan de la Villa de Pasage /

Mode of access: Internet.