Molecular evolution of integrins: Genes encoding integrin β subunits from a coral and a sponge

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin β subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral βCn1 and, especially, the sponge βPo1 sequences are the most divergent of the “β1-class” integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other β subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in βCn1 and βPo1. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.

β-Catenin mutations in cell lines established from human colorectal cancers

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.

The product of ORF O located within the domain of herpes simplex virus 1 genome transcribed during latent infection binds to and inhibits in vitro binding of infected cell protein 4 to its cognate DNA site

The partially overlapping ORF P and ORF O are located within the domains of the herpes simplex virus 1 genome transcribed during latency. Earlier studies have shown that ORF P is repressed by infected cell protein 4 (ICP4), the major viral regulatory protein, binding to its cognate site at the transcription initiation site of ORF P. The ORF P protein binds to p32, a component of the ASF/SF2 alternate splicing factors; in cells infected with a recombinant virus in which ORF P was derepressed there was a significant decrease in the expression of products of key regulatory genes containing introns. We report that (i) the expression of ORF O is repressed during productive infection by the same mechanism as that determining the expression of ORF P; (ii) in cells infected at the nonpermissive temperature for ICP4, ORF O protein is made in significantly lower amounts than the ORF P protein; (iii) the results of insertion of a sequence encoding 20 amino acids between the putative initiator methionine codons of ORF O and ORF P suggest that ORF O initiates at the methionine codon of ORF P and that the synthesis of ORF O results from frameshift or editing of its RNA; and (iv) glutathione S-transferase–ORF O fusion protein bound specifically ICP4 and precluded its binding to its cognate site on DNA in vitro. These and earlier results indicate that ORF P and ORF O together have the capacity to reduce the synthesis or block the expression of regulatory proteins essential for viral replication in productive infection.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Site-specific, photochemical proteolysis applied to ion channels in vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the “signature” Cys128–Cys142 disulfide loop of the nAChR α subunit.

Molding a peptide into an RNA site by in vivo peptide evolution

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial α-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an “RNA world.”

Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein “pocket” binding activity, and do not suppress colony growth of RB(−) cells. In contrast, two low-penetrant alleles (661W and “deletion of codon 480”) retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, “deletion of RB exon 4,” showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(−) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

Phenotype-genotype studies in kuru: Implications for new variant Creutzfeldt–Jakob disease

The PRNP polymorphic (methionine/valine) codon 129 genotype influences the phenotypic features of transmissible spongiform encephalopathy. All tested cases of new variant Creutzfeldt–Jakob disease (nvCJD) have been homozygous for methionine, and it is conjectural whether different genotypes, if they appear, might have distinctive phenotypes and implications for the future “epidemic curve” of nvCJD. Genotype-phenotype studies of kuru, the only other orally transmitted transmissible spongiform encephalopathy, might be instructive in predicting the answers to these questions. We therefore extracted DNA from blood clots or sera from 92 kuru patients, and analyzed their codon 129 PRNP genotypes with respect to the age at onset and duration of illness and, in nine cases, to detailed clinical and neuropathology data. Homozygosity at codon 129 (particularly for methionine) was associated with an earlier age at onset and a shorter duration of illness than was heterozygosity, but other clinical characteristics were similar for all genotypes. In the nine neuropathologically examined cases, the presence of histologically recognizable plaques was limited to cases carrying at least one methionine allele (three homozygotes and one heterozygote). If nvCJD behaves like kuru, future cases (with longer incubation periods) may begin to occur in older individuals with heterozygous codon 129 genotypes and signal a maturing evolution of the nvCJD “epidemic.” The clinical phenotype of such cases should be similar to that of homozygous cases, but may have less (or at least less readily identified) amyloid plaque formation.

Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for ≈64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.

Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.