A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT-CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV40 genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.     

A mutational analysis of the bacteriophage P1 cin recombinase gene: intragenic complementation

Summary Bacteriophage P1 encodes a site-specific recombinase, Cin, which regulates the alternate expression of tail fibre genes by inverting a DNA segment. To define regions of Cin important for the recombination process, we have isolated and characterised 24 different mutations of the cin gene. Most of these mutations affected amino acids that are highly conserved in other related recombinases. Some of these mutants complement each other in vivo. This intragenic complementation could be due to the assembly of heteromers containing both mutant proteins, suggesting that the active enzyme is at least a dimer.     

A detailed multipoint gene map of chromosome 1q

Utilizing genotyping data for 23 markers, we have constructed a 21-locus multipoint genetic map of the long arm of chromosome 1. Five new RFLPs are reported. The map integrates anonymous loci from previous primary linkage maps and incorporates markers for 10 coding sequences. These markers form a continuous linkage group of 85 cM in males and 141 cM in females. The map was constructed employing the LINKAGE and CRIMAP computational methodologies via a stepwise algorithm.     

A mutational analysis of the bacteriophage P1 recombinase Cre

Bacteriophage P1 encodes a 38,600 Mr site-specific recombinase, Cre, that is responsible for reciprocal recombination between sites on the P1 DNA called loxP. Using in vitro mutagenesis 67 cre mutants representing a total of 37 unique changes have been characterized. The mutations result in a wide variety of phenotypes as judged by the varying ability of each mutant Cre protein to excise a lacZ gene located between two loxP sites in vivo. Although the mutations are found throughout the entire cre gene, almost half are located near the carboxyl terminus of the protein, suggesting a region critical for recombinase function. DNA binding assays using partially purified mutant proteins indicate that mutations in two widely separated regions of the protein each result in loss of heparin-resistant complexes between Cre and a loxP site. These results suggest that Cre may contain two separate domains, both of which are involved in binding to loxP.     

Systematic mutational analysis of the yeast beta-tubulin gene.

A systematic strategy was used to create a synoptic set of mutations that are distributed throughout the single beta-tubulin gene of Saccharomyces cerevisiae. Clusters of charged amino acids were targeted for mutagenesis and converted to alanine to maximize alterations on the protein's surface and minimize alterations that affect protein folding. Of the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers heat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that beta-tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yeast at 30 degrees C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most critical for beta-tubulin function. Interestingly, most of the conditional-lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaffected. The exceptions are two mutants that contain abnormally long cytoplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of beta-tubulin mutants and should be valuable in dissecting spindle function.     

Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins.

Expression from a human cytomegalovirus early promoter (E1.7) has been shown to be activated in trans by the IE2 gene products (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281-290, 1989). Using wild-type and mutant viral proteins, we have defined the protein regions required for transactivation of the E1.7 promoter in IE2 and for augmentation of transactivation in the IE1 protein. Two regions of the IE2 proteins were found to be essential for transactivation. One near the amino terminus is within 52 amino acids encoded by exon 3. The second comprises the carboxyl-terminal 85 amino acids encoded by exon 5. The IE2 protein encoded by an mRNA which lacks the intron within exon 5 and the IE2 protein encoded by exon 5 had no activity for transactivation of the E1.7 promoter. Although the IE1 gene product alone had no effect on this early viral promoter, maximal early promoter activity was detected when both IE1 and IE2 gene products were present. The IE1 protein positively regulated its enhancer-containing promoter-regulatory region. The IE1 protein alone increased the steady-state level of IE2 mRNA; therefore, IE1 and IE2 are synergistic for expression from the E1.7 promoter. Like the IE2 proteins, the IE1 protein requires for activity 52 amino acids encoded by exon 3. IE1 also requires amino acids encoded by exon 4. Since the IE1 and IE2 proteins have 85 amino acids in common at the amino-terminal end encoded by exons 2 and 3, the difference between these specific transactivators resides in their carboxyl-terminal amino acids encoded by exons 4 and 5, respectively.     

拟南芥ats1A基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出ats1A基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,ats1A基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

Site-directed mutational analysis of the osmotically regulated proU promoter of Salmonella typhimurium.

We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control. We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect. However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.     

Isolation and structure of a yeast initiator tRNAmet gene.

Sixteen bacterial clones containing yeast initiator tRNAmet genes have been isolated. The size of the BamHI fragments encoding these genes ranges from 4,000 to 23,000 base pairs. The nucleotide sequence of one member of this group has been determined. It has no intervening sequences.