Regions Of Sequence Homology Research Articles

Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4.

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons.

Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

Origin of the chromosome 1 HSR of the house mouse detected by CGH.

The polymorphic Sp100-rs repeat cluster in chromosome band 1D of the house mouse, Mus musculus, makes up as much as 0.1-5% of the haploid genome. 'High-copy' versions of this long-range repeat cluster are cytogenetically apparent as DAPI-negative chromomycin-A3-positive homogeneously staining regions (HSRs). The cluster is a relatively recent acquisition in the genus Mus; the related species M. caroli possesses neither the Sp100-rs cluster nor even the Sp100-rs gene. Except for chromosomes with high-copy clusters, no major rearrangements are visible in chromosomes 1 from M. musculus and M. caroli: they have the same order of G-bands, DAPI-bands and chromomycin A3-bands. Comparative genomic hybridization (CGH) visualizes the cluster in M. musculus and detects a single region of sequence homology to the cluster in M. caroli chromosome band 1D. This indicates that the M. musculus cluster has evolved in situ from sequences originally present in the same chromosome band.

Three period Homologs in Mammals: Differential Light Responses in the Suprachiasmatic Circadian Clock and Oscillating Transcripts Outside of Brain

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows ∼37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

A PCR screen identifies a novel, unconventional myosin heavy chain gene (MYO1) in Tetrahymena thermophila.

Degenerate primers for two regions of sequence homology in the myosin head domain were used in a polymerase chain reaction screen of Tetrahymena thermophila genomic DNA to amplify a 765 bp fragment that was cloned and sequenced. Based on the presence of conserved, myosin-specific sequences, the 765 bp PCR product was identified as a fragment of a myosin gene, the first to be discovered in ciliated protozoa and herein referred to as MYO1. An inverse polymerase chain reaction strategy was used to obtain additional sequence data that included the entire head domain of MYO1. Alignment of the predicted amino acid sequence of the MYO1 head domain with known myosin sequences identified the ATP-binding site, a phosphorylation site, and other myosin-specific consensus regions. In a northern blot analysis, a 765 bp MYO1-specific probe detected a 6.6 kb transcript under highly stringent hybridization conditions. Phylogenetic analysis revealed that the predicted protein encoded by MYO1 is not a member of any of the previously defined myosin classes and therefore represents a presumptive new myosin class.

Analysis of RhoA-binding Proteins Reveals an Interaction Domain Conserved in Heterotrimeric G Protein β Subunits and the Yeast Response Regulator Protein Skn7

To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein beta subunits and in the Saccharomyces cerevisiae Skn7 protein. We show that beta2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.

A regulatory element of the empty spiracles homeobox gene is composed of three distinct conserved regions that bind regulatory proteins

Homeobox genes encode a class of highly evolutionarily conserved transcription factors that control embryonic development. The Drosophila melanogaster empty spiracles gene is the homolog of the two human homeobox genes EMX1 and EMX2. These genes are necessary for central nervous system development. We used a regulatory element of the empty spiracles gene to study the control of homeobox gene expression. The 1.2-kilobase (kb) cis-regulatory element located 3 kb 5' of the transcription start site of the empty spiracles gene was analyzed by evolutionary sequence comparisons, gel mobility shift assays, DNase footprinting, and the generation of transgenic flies. The corresponding element from a related species, Drosophila hydei, was cloned. Three discrete, approximately 100 base pair (bp) regions of sequence homology were identified. Each had two blocks of 10 to 40 bp of near perfect sequence identity. Fusion proteins were produced containing the Abdominal-B homeodomain or the empty spiracles homeodomain, known regulators of empty spiracles gene expression. Gel mobility shift assays showed that each of the three regions is bound by both proteins. DNase footprinting revealed closely linked empty spiracles and Abdominal-B binding sites. We then generated transgenic flies containing a reporter linked to individual conserved regions of the enhancer. Reporter expression was evident only outside of the usual empty spiracles expression domain. These elements are not sufficient alone; a combinatorial model is proposed. Conserved discrete areas within a homeobox gene regulatory element, which function as homeodomain protein transcription factor binding sites, are used in a combinatorial fashion to regulate these developmentally important genes.

The Late Expression Factors 8 and 9 and Possibly the Phosphoprotein p78/83 of Autographa californica Multicapsid Nucleopolyhedrovirus Are Components of the Virus-Induced RNA Polymerase

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its α-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Functional analysis of mammalian per homologues by overexpression systems

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows ∼37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Control of Genes by Mammalian Retroposons

Available data on possible genetic impacts of mammalian retroposons are reviewed. Most important is the growing number of established examples showing the involvement of retroposons in modulation of expression of protein-coding genes transcribed by RNA polymerase II (Pol II). Retroposons contain conserved blocks of nucleotide sequence for binding of some important Pol II transcription factors as well as sequences involved in regulation of stability of mRNA. Moreover, these mobile genes provide short regions of sequence homology for illegitimate recombinations, leading to diverse genome rearrangements during evolution. Therefore, mammalian retroposons representing a significant fraction of noncoding DNA cannot be considered at present as junk DNA but as important genetic symbionts driving the evolution of regulatory networks controlling gene expression.

A Novel GTPase-activating Protein for Rho Interacts with a PDZ Domain of the Protein-tyrosine Phosphatase PTPL1

PTPL1 is an intracellular protein-tyrosine phosphatase that contains five PDZ domains. Here, we present the cloning of a novel 150-kDa protein, the four most C-terminal amino acid residues of which specifically interact with the fourth PDZ domain of PTPL1. The molecule contains a GTPase-activating protein (GAP) domain, a cysteine-rich, putative Zn2+- and diacylglycerol-binding domain, and a region of sequence homology to the product of the Caenorhabditis elegans gene ZK669.1a. The GAP domain is active on Rho, Rac, and Cdc42 in vitro but with a clear preference for Rho; we refer to the molecule as PTPL1-associated RhoGAP 1, PARG1. Rho is inactivated by GAPs, and protein-tyrosine phosphorylation has been implicated in Rho signaling. Therefore, a complex between PTPL1 and PARG1 may function as a powerful negative regulator of Rho signaling, acting both on Rho itself and on tyrosine phosphorylated components in the Rho signal transduction pathway.

EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

A sigma32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity.

sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

Partial reconstitution of a replication-competent retrovirus in helper cells with partial overlaps between vector and helper cell genomes.

Contamination of retroviral vector preparations with replication-competent retroviruses is a major safety concern in human gene therapy. These can arise by recombination between retroviral vectors and packaging cell sequences. Recently, we constructed new packaging lines, derived from spleen necrosis virus (SNV) that do not contain overlapping regions of homology between these two components (DSH134G and DSH29B cells). These cell lines were tested for the presence of recombination products and replication-competent viruses in comparison to a similar packaging line (DSN) that contains a partial overlap between vector and viral protein coding regions. No recombination products were detected in DSH cells. However, we found that recombination had occurred in DSN cells, partially reconstituting a provirus-like structure that was capable of being spread, although inefficiently, through an infected cell culture. Our data indicate that even small regions of sequence homology eventually allow homologous recombination between vector and helper cell genomes.

Genome analysis of filamentous fungi: identification of a highly conserved simple repetitive sequence in different strains of Podospora anserina

A nucleotide stretch located upstream of the MRAS3 gene of the zygomycete Mucor racemosus was found to hybridize to multiple restriction fragments of genomic DNA of the ascomycete Podospora anserina. DNA of strains derived from different geographical locations revealed no major restriction length fragment polymorphisms. Also, no age-related polymorphisms were observed. The repetitive sequence is located on all of the physically fractionated Podospora chromosomes. Sequence analysis of the upstream sequence of MRAS3 and of a DNA-stretch from a genomic clone of P. anserina which hybridized to the Mucor probe revealed a region of sequence homology. This sequence is composed of alternating G and T residues [poly(dG-dT) or poly(GT)] of 76 bp in length and thus represents a simple repetitive sequence, or microsatellite. A synthetic oligonucleotide, (GT) 8, corresponding to this sequence gave also rise to a complex hybridization pattern. Screening of genomic libraries of P. anserina revealed poly(GT)-stretches on about 5–7% of all clones. Finally, this repetitive sequence was identified in the genome of the filamentous fungi Sordaria macrospora and Curvularia lunata.

The chicken βA4- and βB1-crystallin-encoding genes are tightly linked

Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4-crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1-cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens-specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1-cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma-cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens.

Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity.

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences in the second internal transcribed spacer (Ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae)

The second internal transcribed spacer (ITS-2) of the ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3–7.6%), with the species infecting birds ( T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichotrongyle nematodes.

Dissection of functional domains in Bcl-2 alpha by site-directed mutagenesis.

Bcl-2 alpha is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2 alpha or Bcl-2 beta, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2 alpha is active and membrane bound, but Bcl-2 beta is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane association of Bcl-2 alpha. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overexpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2 alpha/beta splice site, as well as part of domain X in Bcl-2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)