Bruno Luckow

CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements

The coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene is wide1y used as an indicator gene in gene transfer experiments dealing with regulation of transcription in eukaryotes. Chimaeric CAT fusion genes are especially useful because no endogenous CAT activity is present in eukaryotic ce11s and because CAT enzyme activity can be monitored by a rapid and sensitive assay (1). In order to simplify the construction of hybrid CAT genes, we have constructed the plasmids pBLCA T2 and pBLCAT3. The coding region of the CA T gene as well as the small t intron and polyadenylation signals from SV40 were inserted into the polylinker region of the high copy number plasmid pUC18 (2). Unique BglII and XhoI restrietion sites were introduced upstream of the CAT coding region by insertion of synthetic linkers. A BamHI site at the 3 end of the transcription unit was converted into adam methylation sensitive ClaI site by partial digestion with BamHI, filling in and re -ligation. In the promoterless construction pBLCAT3 eight unique restriction sites are suitable for insertion of different eukaryotic promoters at the 5 end of the CAT gene. Four additional unique restriction sites rnake the insertion of regulatory signals 3 of the CAT gene possible and enable the excision of the intact fusion gene from the prokaryotic vector. The presence of the Herpes simplex virus tk promoter in pBLCAT2 permits the analysis of the effects of putative regulatory elements on a heterologous eukaryotic promoter. A BamHIIBgllI fragment from the HSV tk linker scanning mutant LS 115/ 105 (3) spanning the promoter from 105 to + 51 was inserted into the corresponding restriction sites of pBLCAT3 thereby generating pBLCAT2. The modified polylinker regions at the 5 and the 3 ends have been sequenced and compiled sequences for both plasmids are available on request.

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation‐dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

Reporter constructs with low background activity utilizing the cat gene

Reporter plasmids utilizing the cat gene for the analysis of promoter and enhancer sequences in vertebrate cells, were constructed. These plasmids minimize the background of transcription derived from cryptic promoters or cryptic regulatory elements within the vector.

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).