Siegfried Ruppert

Functional analysis of alternatively spliced tyrosinase gene transcripts.

Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c‐locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.

Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors

Using in vivo dimethylsulfate footprinting, we have analyzed protein-DNA interactions within two regions upstream of the tyrosine aminotransferase (TAT) gene that are characterized by an altered chromatin structure in TAT-expressing as compared to nonexpressing cells. All the identified protein contacts to DNA are found exclusively in the TAT-expressing hepatoma cells. In vitro analyses of specific DNA-binding factors in crude nuclear extracts yield DNAase I footprints that correlate well with the binding sites in vivo. Surprisingly, all DNA-binding activities are present in nuclei of TAT-expressing and nonexpressing cells, indicating that the mere presence of factors is not sufficient for their interaction with a binding site in vivo. Genomic sequencing reveals methylation of CpG dinucleotides in the regions analyzed in nonexpressing cells, whereas no methylation is found in TAT-expressing cells. In vitro methylation at a cytosine residue within a footprint region prevents the interaction of a factor with its binding site.

TAFII250 Is a Bipartite Protein Kinase That Phosphorylates the Basal Transcription Factor RAP74

Some TAF subunits of transcription factor TFIID play a pivotal role in transcriptional activation by mediating protein-protein interactions, whereas other TAFs direct promoter selectivity via protein-DNA recognition. Here, we report that purified recombinant TAFII250 is a protein serine kinase that selectively phosphotylates RAP74 but not other basal transcription factors or common phosphoacceptor proteins. The phosphorylation of RAP74 also occurs in the context of the complete TFIID complex. Deletion analysis revealed that TAFII250 contains two distinct kinase domains each capable of autophosphorylation. However, both the N- and C-terminal kinase domains of TAFII250 are required for efficient transphosphorylation of RAP74 on serine residues. These findings suggest that the targeted phosphorylation of RAP74 by TAFII250 may provide a mechanism for signaling between components within the initiation complex to regulate transcription.

Multiple mRNA isoforms of the transcription activator protein CREB: Generation by alternative splicing and specific expression in primary spermatocytes

We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE‐binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway.

Multiple transcripts of the mouse tyrosinase gene are generated by alternative splicing.

We report the cloning and isolation of the mouse tyrosinase cDNA by screening mouse B16 melanoma cDNA libraries. Independent cDNA clones have been characterized by restriction enzyme analysis, hybridizations with individual subprobes and by partial sequencing analysis. Based on these criteria we have identified multiple transcripts, which in comparison to the major transcript, display deletions of internal sequences and have different 3′ termini. The most abundant transcript encodes a functional tyrosinase. The structural gene which encodes five exons separated by large introns and spans a chromosomal region of approximately 70 kb has been isolated. Comparison of the cDNAs with the cloned genomic DNAs and sequencing of the exon/intron boundaries reveal that the multiple transcripts are generated by alternative splicing and putatively by alternate polyadenylation site usage. The alternative splicing mechanisms involve exon skipping as well as internal donor splice site usage. Primer extension analysis shows that the transcripts are produced from two different promoters. Southern blot analysis of DNAs derived from mice carrying the lethal albino deletion mutations demonstrates that the structural gene maps near or at the albino locus. The viable albino mouse BALB/c carries an apparently intact structural gene indicating that the albino phenotype is a consequence of a failure to express the tyrosinase gene or the inability to produce a tyrosinase enzyme.

Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice

The c‐locus of the mouse is thought to encode tyrosinase, the key enzyme for melanin synthesis in melanocytes of the skin and the eye. Recently, a mouse cDNA was isolated and shown to confer tyrosine activity on a cell line which expressed no specialized functions for melanin synthesis. To verify that the isolated tyrosinase gene is encoded at the genetically well characterized c‐locus, a minigene was assembled from tyrosinase cDNA and tyrosinase genomic DNA and used for generation of transgenic mice. Following microinjection of this construct into fertilized eggs of an albino mouse strain, transgenic mice were obtained which showed pigmentation in skin and eyes. By in situ hybridization, we show expression of the transgene in melanocytes of the hairbulb and in the pigmented cell layers of the eye. We conclude that we have rescued the albino mutation (c/c) by introduction and expression of a functional tyrosinase gene.

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

Mice homozygous for deletions around the albino locus fail to activate expression of a set of neonatal liver functions and die shortly after birth. This phenotype is thought to result from the loss of a positive transacting factor, denoted alf, in deletion homozygotes. Using differential cDNA screening, we isolated and characterized genes whose cell type-specific transcription is affected by alf and found as a common feature that expression of these genes is induced by glucocorticoids and cAMP. Surprisingly, a subset of these alf-responsive genes is negatively controlled by the tissue-specific extinguisher locus Tse-1. Administration of glucocorticoids and cAMP leads to reversal of Tse-1-mediated extinction of these genes. These results show that two trans-acting factors coordinately regulate expression of overlapping sets of liver-specific genes. We suggest that both the lethal phenotype and the extinguished state result from interference with hormone signal transduction.

Cloning and expression of Drosophila TAFII60 and human TAFII70 reveal conserved interactions with other subunits of TFIID.

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter‐specific activators while others serve as interfaces for TAF‐TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved.

The mouse CREB (cAMP responsive element binding protein) gene: Structure, promoter analysis, and chromosomal localization

In this paper we report the isolation and characterization of the mouse CREB gene. It is composed of 11 exons and 10 introns and spans a region of 70 kb. BR-A and BR-B, the two alpha-helical regions of the proposed basic DNA binding domain of CREB, are encoded separately on exons 10 and 11. The mouse CREB gene is expressed from a promoter that is situated in a CpG island. The promoter contains no TATA or CCAAT box homologies but has a number of putative binding sites for the acidic transcriptional activator Sp1 and a 9/11 match with the initiator region. Transcriptional start site mapping identified five major start sites spread over at least 41 nucleotides. Northern blot analysis indicated that expression of the CREB gene is almost ubiquitous with expression at differing levels of multiple transcripts. Testis expressed a predominant RNA species of approximately 1.6 kb. The CREB gene was found to be single copy in the mouse and well conserved through evolution. Finally Creb-1, the CREB locus, was mapped to the proximal region of mouse chromosome 1.

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.