Regulation of apoptosis by BH3 domains in a cell-free system.

BACKGROUND: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins. RESULTS: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system. CONCLUSIONS: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.

A human aldehyde dehydrogenase (aldose reductase) pseudogene: nucleotide sequence analysis and assignment to chromosome 3.

Four cosmid clones containing putative pseudogenes for human aldehyde dehydrogenase (Aldose reductase) were isolated from libraries made to two individuals. These clones show different patterns on digestion with restriction endonucleases and probably represent distinct and separate loci. The DNA sequence of one of the putative pseudogenes (cosmid AR.F) was determined, and comparisons demonstrate 89.7% homology with the cDNA sequence of the functional aldose reductase gene. This pseudogene sequence contains no intronic sequences, whereas the functional aldose reductase has nine introns. In addition, the homology disappears in region 5' to the transcription start site for the cDNA, implying that regulatory elements such as the promoter are missing from this pseudogene. The pseudogene defined by cosmid AR.F has been mapped to chromosome 3 by polymerase chain reaction using amplimers specific for this pseudogene to amplify DNA from somatic cell hybrids.

Structure of the human aldose reductase gene.

The structure and sequence of the human gene for aldose reductase (AR) was determined by analysis of cDNA and genomic clones. The AR gene was independently isolated from two different cosmid libraries and the clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene extends over approximately 18 kilobases and consists of 10 exons giving rise to a 1,384 nucleotide mRNA (excluding the poly(A) tail). The human aldose reductase gene codes for a 316-amino acid protein with a molecular mass of 35,858 daltons. The size range for the exons is 82-168 base pairs (bp), whereas that for the introns is 325 to about 7,160 bp. A major site of transcription initiation in liver was mapped to an A residue 31 nucleotides upstream from the A of the ATG initiation codon. The promotor region of the gene contains a TATA (TATTTA) box and a CCAAT box which are located 37 and 104 nucleotides upstream, respectively, from the transcription initiation site. We have found four Alu elements in the AR gene; two are found in intron 1 and one each in intron 4 and intron 9.

DNA sequence analysis of the KM19 locus linked to cystic fibrosis. Design of new oligonucleotides to remove non-specific PCR products.

The PstI polymorphism detected by probe KM19 is a highly informative marker in linkage disequilibrium with the cystic fibrosis locus and has been used extensively for prenatal diagnosis. The currently available primers used for polymerase chain reaction- (PCR-) based analysis of this locus have been shown to produce spurious amplification products. In this report, we describe the sequence of the KM19 locus and the major contaminating PCR product. We have used this information to design a more specific amplification procedure for analysis of the KM19 locus.

Multiple tandem 18-kb sequences clustered in the region of the acute promyelocytic leukemia breakpoint on chromosome 17.

This paper describes the cloning of an 18-kb sequence present in approximately 30 copies on chromosome 17. Most of these are clustered in the region of the breakpoint associated with acute promyelocytic leukemia (APL). These copies map both above and below the breakpoint, and pulsed field gel analysis indicates that the majority of these sequences lie within a region of approximately 2 megabases. The organization of these sequences appears to be that of large imperfect palindromes.

Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer.

We used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for greater than 20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization, we propose the following order for these loci: pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z 1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UM PH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, we have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.

Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9.

Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed. HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site.

Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein.

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.

Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin.

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.

A gene fusion that localises the penicillin-binding domain of penicillin-binding protein 3 of Escherichia coli.

A gene fusion that links the COOH-terminal 349 amino acids of penicillin-binding protein 3 (60 kDa) of E. coli to the NH2-terminus of beta-galactosidase has been constructed. The fusion protein (38.5 kDa) retains the ability to bind benzylpenicillin with high affinity, establishing that the penicillin-binding domain (and presumably the penicillin-sensitive transpeptidase activity) of this high molecular mass penicillin-binding protein is located on a COOH-terminal functional domain.