Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays.

The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high-density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence-induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.

Quantitative expression analysis of the small conductance calcium-activated potassium channels, SK1, SK2 and SK3, in human brain.

Small conductance calcium-activated potassium (SK) channels are important in controlling neuronal excitability and three SK channels have been identified to date. In the present study, we report the first quantitative analysis of SK1, SK2 and SK3 expression in human brain using TaqMan RT-PCR on a range of human brain and peripheral tissue samples. SK1 expression is restricted to the brain whereas SK2 and SK3 are more widely expressed.

Expression patterns of zebrafish sox11A, sox11B and sox21.

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.

Physical mapping at 6q27 of the locus for the TATA box-binding protein, the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB, strongly suggests that it is single copy in the human genome.

The TATA box-binding protein (TBP) has a fundamental role in eukaryotic cell metabolism, since it is necessary for transcription of class I, class II, and class III genes; in fact, TBP is the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB. Contrary to the previously hypothesized existence of a family of genes coding for DNA-binding proteins highly related to TBP, our experiments show that the segment coding for the evolutionarily well-conserved carboxyl-terminal domain, involved in DNA binding, is unique; accordingly, we conclude that the TBP locus itself, which we have localized to 6q27, is single copy in the human genome. On the other hand, a cDNA fragment coding for the evolutionarily variable amino-terminal domain detects multiple cross-hybridizing sequences in the genome of higher eukaryotes. We suggest that the common motif is represented by the long string of glutamine codons, which characterizes the amino-terminal segment of human TBP: in fact, other proteins involved in transcription, such as TAF II 110, Sp1, and some homeobox proteins, are known to contain glutamine-rich segments.

The gene for SP-40,40, human homolog of rat sulfated glycoprotein 2, rat clusterin, and rat testosterone-repressed prostate message 2, maps to chromosome 8.

Sulfated glycoprotein 2 (SGP-2) is a rat glycoprotein that is particularly abundant in seminal fluid, where it is found associated with the acrosome and the tail of mature spermatozoa; for this reason it has been suggested that it has an important role in spermatogenesis. On the basis of nucleotide sequence homology, it has been proposed that the orthologous human gene is that coding for serum protein-40,40 (SP-40,40), a serum protein also called complement lysis inhibitor (CLI), SP-40,40 has been shown to act as a control mechanism of the complement cascade: in fact, it prevents the binding of a C5b-C7 complex to the membrane of the target cell and in this way inhibits complement-mediated cytolysis. SGP-2 and SP-40,40 seem then to be part of different biological systems. Furthermore it has been shown that another protein, testosterone-repressed prostate message 2 (TRPM-2), shares sequence homology with SGP-2 and SP-40,40. TRPM-2 is expressed at high levels and in a temporally precisely defined manner in dying cells, an observation that would suggest its involvement in the cascade of events leading to cell death. We have used a large panel of 24 mouse/human hybrid cell lines and a cDNA for SGP-2, which is also highly homologous to that for rat clusterin, to map the chromosomal location of the orthologous human gene. The mapping data and the Southern analysis presented in this paper, in addition to the data available from the literature, strongly suggest that in the human genome there is a single locus homologous to the probe used and that it codes for the protein which has been called, in different species, SP-40,40, SGP-2, clusterin, and TRPM-2. The chromosomal mapping of the locus for this multiname protein should facilitate its cloning and a better understanding of the apparently many biological functions of its product.