The mouse Pgk-1 gene promoter contains an upstream activator sequence

Abstract

The Pgk-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed. This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells. The Pgk-1 promoter is in a region rich in nucleotides G and C. This promoter can efficiently drive high levels of expression of reporter genes such as E. coli lacZ and neo. We have determined that the 120 bp upstream of the transcription start site functions as a core promoter. Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion. This 320 bp region does not enhance transcription from the core promoter of the SV40 early region. Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity.

Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation

“…The EF1a promoter is highly active in stimulating transcription of heterologous genes in a variety of mammalian cell lines (Mizushima and Nagata, 1990). Cells were cotransfected with pEBB-PU.1 and pPGKneo (McBurney et al, 1991) by lipofection and transfectants were selected in Geneticin (G418). We also cotransfected MEL cells with pPGKneo and a construct in which the PU.1 cDNA was placed in the antisense (AS) orientation relative to the EF1a promoter.…”

“…For DNA transfection, exponentially growing cells were centrifuged, washed twice in DME medium, and resuspended at a density of 10 7 cells/ml in DME medium. 75 mg of pEBB-PU.1 or pEBB-AS-PU.1 and 0.5 mg of pPGKneo (McBurney et al, 1991) were mixed in 0.3 ml DME medium and added to 0.3 ml DME medium containing 50 ml of lipofectin (GIBCO-BRL). The DNA-lipid mixture was incubated for 15 min at room temperature, and then added to 0.5 ml of cell suspension in a cell culture¯ask.…”

Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1.

“…The PGK-neo gene was excised from PGK-neo-bpA (McBumey et al 1991) by digestion with Xhol and cloned into the Xhol site of Bluescript II KS(+) (Stratagene). This subclone was cut with BamHl and Hindlll and ligated to a 6.2-kb BamHl-Hindlll frag ment of the mCAT-1 genomic locus (comprising the 5' homol ogy) containing coding exons 2 and 3.…”

An Upstream Control Region Required for Inducible Transcription of the Mouse H1° Histone Gene during Terminal Differentiation

“…The presence of a small foreign sequence in the mRNA produced from the transfected gene allows it to be distinguished from the endogenous H1Њ mRNA when an antisense RNA probe prepared from the marked gene is used in RNase protection assays. The marked H1Њ gene construct was ligated to a plasmid containing the selectable marker gene PGK-neo (54), allowing selection of transfected cells with G418. Since integration of the transfected DNA at different sites in the genome of transfected cells often leads to large variations in the level of expression and inducibility of a gene, several large pools of transfectants, each consisting of at least 200 clones, were prepared for each construct.…”