Identification and characterization of the potential promoter regions of 1031 kinds of human genes.

To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the "oligo-capping" method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA(+)/Inr(+) PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and the TM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

Diverse transcriptional initiation revealed by fine, large-scale mapping of mRNA start sites.

Determination of the mRNA start site is the first step in identifying the promoter region, which is of key importance for transcriptional regulation of gene expression. The 'oligo-capping' method enabled us to introduce a sequence tag to the first base of an mRNA by replacing the cap structure of the mRNA. Using cDNA libraries made from oligo-capped mRNAs, we could identify the transcriptional start site of an individual mRNA just by sequencing the 5'-end of the cDNA. The fine mapping of transcriptional start sites was performed for 5880 mRNAs in 276 human genes. Contrary to our expectations, the majority of the genes showed a diverse distribution of transcriptional start sites. They were distributed over 61.7 bp with a standard deviation of 19.5. Our finding may reflect the dynamic nature of transcriptional initiation events of human genes in vivo.

Transmembrane-domain trapping: a novel method for isolation of cDNAs encoding putative membrane proteins.

We have developed a method that enables us to isolate cDNAs of putative membrane proteins. The system is designed to isolate a cDNA which can provide the transmembrane domain to the extracellular part of the IL-2 receptor alpha chain. We constructed a p18Mac vector by putting part of the IL-2 receptor alpha chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRalpha. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor alpha chain will be expressed on the surface of the transfected cells. Otherwise, the chimeric protein will be either secreted or retained inside the transfected cells. We made a cDNA library using p18Mac and screened for cDNA clones which allowed the expression of the extracellular domain of the IL-2 receptor alpha chain on the cell surface. Of the 2000 clones screened, 5 clones were scored as positive. Partial sequence analysis revealed that one clone encoded the amyloid precursor protein, two others encoded mitochondrial proteins and the rest were new. These results suggest the system is effective in isolating cDNAs encoding putative membrane proteins.

Use of an epitope-tagged cDNA library to isolate cDNAs encoding proteins with nuclear localization potential.

We have combined epitope tagging with an expression cDNA library in order to isolate cDNAs encoding nuclear proteins. This system allows us to detect proteins expressed from the cDNA library by using antibodies against the epitope tag. As a tag, we used the 85-aa N-terminal peptide of the SV40 T antigen which lacks the nuclear localization signal (NLS). A strong expression vector, pEF204 [Kim et al., Gene 91 (1990) 217-223], was modified into an epitope-tagging vector, pTkim, by putting the tag-coding region and a cDNA cloning site immediately after its promoter. From cDNA libraries constructed using pTkim, we isolated eight cDNA clones whose tagged proteins were localized within the nuclei. From partial sequence analysis, two cDNAs were shown to code for the ribosomal (r-) proteins, simian L44 and human L21, and the others were shown to be new. Furthermore, six cDNAs including those encoding the r-proteins could direct a non-karyophilic T antigen [Fischer-Fantuzzi et al., Virology 153 (1986) 87-95] into nuclei, showing that they have NLSs. These results indicate that this system is useful for isolating new cDNAs which code for nuclear proteins.

Cell-type-specific transcription of an immunoglobulin kappa light chain gene in vitro.

We have established a cell-free system, derived from a human B-cell lymphoma, in which immunoglobulin kappa light chain gene promoters are both accurately transcribed and regulated in a cell-type-specific manner. Thus, accurate transcription from the T1 kappa light chain gene promoter was much more efficient in B-cell extracts than in HeLa cell extracts, whereas control promoters (adenovirus major late and histone H2B) were transcribed equally well in either extract. More important, the increased kappa light chain gene transcription in B-cell extracts was dependent upon upstream sequences (containing the conserved decanucleotide element) previously shown to be necessary for B-cell-specific transcription in vivo; in contrast, removal of these sequences had no effect on the low level of kappa transcription in HeLa extracts. The maximal level of upstream sequence-mediated transcription was dependent upon template topology. These studies show that there is at least one B-cell-specific factor that stimulates transcription from purified DNA templates, and they further suggest that the in vivo action of the factor(s) on other components of the transcription machinery is direct rather than indirect (e.g., via the maintenance of an open chromatin structure). The cell-free system described here should facilitate both purification and functional studies of the B-cell-specific factor(s).

Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control.

We have located the DNA sequence involved in the stringent control of the Escherichia coli tufB operon. Various deletion and insertion mutants of the promoter locus were constructed by in vitro mutagenesis, and their response to guanosine-5'-diphosphate-3'-diphosphate (ppGpp) was examined in a cell-free transcription system consisting of purified RNA polymerase holoenzyme. The nucleotide sequence (GpCpGpC) from positions -7 to -4 (designating the initiation site of mRNA as position +1) is responsible for the selective inhibition by ppGpp of tufB transcription. Point mutations were then constructed in which each one of the above four nucleotides was replaced by an A or T residue and tested for their response to ppGpp in the in vitro transcription system. The results indicated that the alteration of any nucleotide in the GpCpGpC sequence leads to the loss of the stringent response.

Selective inhibition of transcription of the E. coli tufB operon by guanosine-5'-diphosphate-3'-diphosphate.

We have studied the effect of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) on the transcription of the E. coli tufB and recA operons in a cell-free system containing of purified RNA polymerase holoenzyme. The transcription of the tufB operon which is under stringent control, was markedly inhibited by 0.5 mM ppGpp, and the extent of this inhibition was found to be greatly influenced by the Mg2+ and K+ concentrations in the reaction mixture. Maximal inhibition was obtained in the presence of 2 mM Mg2+ and 80-120 mM K+, whereas at higher concentrations of Mg2+ or lower concentrations of K+, practically no inhibition was observed. In contrast, transcription of the recA operon which is not subject to stringent control, was little affected by ppGpp at any of Mg2+ and K+ concentrations tested. The nucleotide inhibited initiation of transcription of tufB, while the rate of RNA chain elongation was not greatly inhibited in the presence of ppGpp.

Flexural rigidity of singlet microtubules estimated from statistical analysis of their contour lengths and end-to-end distances.

Microtubules in solutions, observed under a dark-field microscope, show incessant Brownian movement such as translational, rotational and flexing motion. A large number of microtubules, spontaneously stuck to the under surface of a coverslip, were photographed and the contour lengths and end-to-end distances of their images were measured. From the statistical analysis of the contour lengths and end-to-end distances, a value for the parameter lambda representing the flexibility of singlet microtubules was estimated to be lambda = (6.8 +/- 0.8) . 10(-3) micrometers-1. From the value of lambda, the elastic modulus for bending, epsilon, and Young's modulus, Y, of singlet microtubules were computed to be epsilon = approximately 10(-16) and Y = approximately 10(9) dyne . cm-2, respectively. The microscopic elastic constant, k, of bonding between two tubulin monomers neighboring along the singlet microtubule was computed to be k = congruent to 10(2) dyne . cm-1. A singlet microtubule is an order of magnitude as strong against bending and as weak against stretching as an F-actin filament.