Two distinct factors interact with the promoter regions of several liver-specific genes.

A segment of the human alpha 1-antitrypsin (alpha 1AT) 5'-flanking region comprising nucleotides -137 to -37 from the start of transcription is sufficient to drive liver-specific transcription from the homologous alpha 1AT promoter and from the heterologous SV40 promoter. In this paper we characterize two proteins, LF-A1 and LF-B1, whose ability to bind wild-type and mutant alpha 1AT promoter segments correlates with the ability of these segments to activate transcription in vivo. DNase I protection and methylation interference analysis reveals that LF-A1 recognizes sequences present in the regulatory region of the human alpha 1-antitrypsin, apolipoprotein A1 and haptoglobin-related genes. These sequences share a common 5' TGG/A A/C CC 3' motif. LF-B1 binds to the palindrome 5' TGGTTAAT/ATTCACCA 3' which is present in the human alpha 1-antitrypsin gene between positions -78 and -62 from the start of transcription. LF-B1 also recognizes a related sequence present in the human albumin gene between -66 and -50. These results suggest that LF-A1 and LF-B1 are common positive trans-acting factors which are required for the expression of several genes in the hepatocyte.

The transcription factor LF-A1 interacts with a bipartite recognition sequence in the promoter regions of several liver-specific genes.

The transcription factors LF-A1 and LF-B1 are required for the cell-specific expression of the human alpha 1-antitrypsin gene in hepatocytes. We report here the purification and preliminary characterization of LF-A1. This protein, purified to homogeneity from calf liver nuclei by site-specific DNA affinity chromatography and reverse-phase HPLC, has a molecular mass of 40 kDa. Binding sites of LF-A1 are present in the promoter regions of several genes expressed in the liver (alpha 1-antitrypsin, apolipoproteins A1, B1, A4 and pyruvate kinase). Interestingly, the binding site of LF-A1 is bipartite and consists of two short sequence motifs (consensus: TGGACT/CT/C and TGGCCC) separated by a variable 'spacer' region. Insertion or deletion of 1-4 nucleotides in the 'spacer' region of the site in the alpha 1-antitrypsin promoter does not abolish DNA binding.

Self-cloning in the cyanobacterium Anacystis nidulans R2: fate of a cloned gene after reintroduction.

Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self-cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid.