We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady‐state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462‐amino‐acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.
We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.
One hallmark of Alzheimer disease is the formation in the brain of amyloid plaques containing a small peptide derived from the fi-amyloid precursor protein (APP).The APP gene exhibits a complex pattern of expression in peripheral tissues and in the brain. The entire human APP gene was introduced into embryonic stem (ES) cells by co-lipofection of a 650-kb yeast artificial chromosome (YAC). Three ES lines containing an essentiafly intact YAC were isolated, and expression of human APP mRNAs at levels comparable to those of endogenous mouse APP transcripts was obtained. A transgenic mouse line was established by germ-line transmission of the APP YAC. RNase protection analysis of human APP mRNAs demonstrated appropriate splicing of the primary APP transcript in ES cells and in the brain of a transgenic animal. These mice may be useful for elucidating the function of the various APP isoforms in vivo.The senile plaques characteristic of Alzheimer disease (AD) consist of abnormal neurites around an amyloid core primarily composed of a 4-kDa polypeptide, /3-amyloid. The 3-amyloid peptide is derived from the cellular processing of ,B-amyloid precursor protein (APP). Mutations in the APP gene have been identified in certain cases of familial AD, suggesting a causal role for ,B-amyloid deposition in at least some forms of AD (1, 2). The human APP gene is located on chromosome 21, and Down syndrome (DS) individuals overexpress the wild-type APP protein (3). Those who survive past their thirties invariably develop an AD neuropathology (4), and immunoreactive f-amyloid depositions can be found prior to any detectable neuronal degeneration in the DS brain (5). Although APP is expressed at normal levels in non-DS cases of AD, the phenotype seen in DS individuals suggests that AD may be initiated or accelerated by overexpression of APP.The APP gene is composed of 18 exons distributed over several hundred kilobases (6). Alternative splicing of the primary transcript results in the production of at least five distinct transcripts. Of the three predominant APP isoforms, APP770 and APP751 contain a Kunitz-type protease inhibitor (KPI) domain, and are found at varying levels in many tissues (7,8). APP695, lacking the KPI domain, is the predominant splice form in the brain and is less abundant in the periphery (7). Other APP variants have been detected at lower levels (9-11). A possible role for the various isoforms in the progression of AD is suggested by changes in AD brains of the ratio of the KPI-containing APP variants to . However, it is clear that the APP gene exhibits complex tissue-and cell type-specific expression in human (10) and mouse (16) brain.The lack of a small-animal model for AD has hampered investigation of the early events in the progression of AD.
We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response.
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