43K Protein Research Articles

Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm.

Secretions of the uterus support survival and growth of the conceptus (embryo/fetus and associated membranes) during pregnancy. Galectin-15, also known as OVGAL11 and a previously uncharacterized member of the galectin family of secreted beta-galactoside lectins containing a conserved carbohydrate recognition domain and a separate putative integrin binding domain, was discovered in the uterus of sheep. In endometria of cyclic and pregnant sheep, galectin-15 mRNA was expressed specifically in the endometrial luminal epithelium but not in the conceptus. In pregnant sheep, galectin-15 mRNA expression appeared in the epithelia between days 10 and 12 and increased between days 12 and 16. Progesterone induced and IFN-tau stimulated galectin-15 mRNA in the endometrial epithelium. Galectin-15 protein was concentrated near and on the apical surface of the endometrial luminal epithelia and localized within discrete cytoplasmic crystalline structures of conceptus trophectoderm (Tr). In the uterine lumen, secreted galectin-15 protein increased between days 14 and 16 of pregnancy. Galectin-15 protein was functional in binding lactose and mannose sugars and immunologically identical to the unnamed Mr 14,000 (14K) protein from the ovine uterus that forms crystalline inclusion bodies in endometrial epithelia and conceptus Tr. Based on the functional studies of other galectins, galectin-15 is hypothesized to function extracellularly to regulate Tr migration and adhesion to the endometrial epithelium and intracellularly to regulate Tr cell survival, growth, and differentiation. Galectins may be useful as cellular and molecular markers for endometrial function and receptivity, to enhance conceptus survival and development, and to evaluate and enhance fertility.

Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line.

The human adenovirus E4 ORF 6 34 kDa oncoprotein (E4 34k), in concert with the 55 kDa product of E1b, prevents concatenation of viral genomes in infected cells, inhibits the repair of double strand breaks (DSBs) in the viral genome, and inhibits V(D)J recombination in a plasmid transfection assay. These activities are consistent with a general inhibition by the E4 34k and E1b 55k proteins of DSB repair by non-homologous end joining (NHEJ) on extrachromosomal substrates. To determine whether inhibition of NHEJ extends to repair of DSBs in the cell chromosome, we have examined the effects of E4 34k on repair of chromosomal DSBs induced by ionizing radiation in a cell line in which E4 34k expression and biological activity is inducible and E1b 55k is produced constitutively. We demonstrate that in this cell line, induction of E4 34k inhibits chromosomal DSB repair. Recently, it has been shown that in infected cells, E4 34k and the adenovirus E1b 55k proteins cooperate to destabilize Mre11 and Rad50, components of mammalian NHEJ systems. Consistent with this, induction of expression of E4 34k in the inducible cell line also reduces the steady state level of Mre11 protein.

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein.

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.

Stability in vitro of the 69K movement protein of Turnip yellow mosaic virus is regulated by the ubiquitin-mediated proteasome pathway.

Plant viruses move to adjacent cells with the use of virus-encoded cell-to-cell movement proteins. Using proteins produced by in vitro translation, we present evidence that the '69K' movement protein of Turnip yellow mosaic virus (TYMV) is recognized as a substrate for the attachment of polyubiquitin chains and for subsequent rapid and selective proteolysis by the proteasome, the ATP-dependent proteolytic system present in reticulocyte lysate. Truncation of the 69K protein suggests the existence of two degradation signals within its sequence. We propose that selective degradation of virus movement proteins may contribute to the previously reported transient nature of their accumulation during infection.

SPARC expression in primary human renal cell carcinoma: Upregulation of SPARC in sarcomatoid renal carcinoma

SPARC (secreted protein acidic and rich in cysteine, also called osteonectin, BM-40, and 43K protein) is a matricellular protein and is associated with cell–matrix interactions during cell proliferation and extracellular remodeling. It is also implicated in the neovascularization, invasion, and metastasis of human malignancies. To investigate a potential role of the SPARC in renal tumorigenesis, we examined primary renal cell carcinomas (RCCs) for SPARC expression by Northern blot analysis and for protein distribution by immunohistochemistry. We found that 6 (100%) of 6 sarcomatoid and 25 (70%) of 36 clear-cell carcinomas had enhanced SPARC transcription compared with that of the corresponding normal kidney tissue. In contrast, papillary and chromophobe RCCs characterized by a hypovascular or avascular tumor phenotype had undetectable SPARC expression. Immunohistochemical analysis showed that SPARC was strongly stained in the cytoplasm of the sarcomatoid neoplastic cells in sarcomatoid RCCs, whereas it was expressed only in the vascular endothelial cells and fibroblasts in clear-cell RCCs. SPARC staining intensity in the stromal cells was increased in the invading portion in some clear-cell RCCs. These findings suggest that tumor development, including neovascularization and invasion in clear-cell RCCs, might be regulated by SPARC from stromal endothelial cells and fibroblasts and that sarcomatoid transformation from common-type RCCs is associated with upregulation of SPARC expression; SPARC may contribute to its aggressive tumor phenotype. HUM PATHOL 32:1064-1070. Copyright © 2001 by W.B. Saunders Company

Vaccines for Moraxella catarrhalis

Vaccine development for Moraxella catarrhalis is in the antigen identification stage. M. catarrhalis does not appear to synthesize secreted antigens such as exotoxins, nor does it appear to possess a carbohydrate capsule. Modified forms of these antigens are usually good vaccine components. There is some interest in whole bacterial cells and membrane fractions, but the search has largely focused on purified outer surface antigens. All of the present antigens have been selected based on the response seen in animals, although the antibody response seen in people exposed to the bacterium provides some guidance. The antibody response provides information related to the cross-strain preservation of epitopes and whether they are surface exposed. Antigens that elicit antibodies that have complement dependent bactericidal capacity, opsonophagocytic activity or interfere with one of the antigen's known functions such as adhesion or nutrient acquisition are particularly valued. In addition to examining the antibody response, some antigens have been evaluated in a murine pulmonary clearance model. Using these assays and model, several vaccine candidates have been identified. The antigens may be roughly classified by the function they serve the bacterium. One set appears to promote adhesion to host tissues and includes the hemagglutinins, ubiquitous surface protein A1 (UspA1), and possibly the CD protein. A second set is involved in nutrient acquisition. This set includes the lactoferrin binding protein A (LbpA) and lactoferrin binding protein B (LbpB), the transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB), the CD and E porins, and the Catarrhalis outer membrane protein B (CopB). A third set is comprised of antigens involved in virulence and it includes lipooligosaccharide (LOS) and the ubiquitous surface protein A2 (UspA2). Antigens of unknown function, such as the 200K protein, may also be vaccine candidates. The antigens that are most suitable will be determined in clinical studies that are only beginning now.

Genetic Analysis of a Potential Zinc-binding Domain of the Adenovirus E4 34k Protein

E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral DNA replication, apoptosis, double strand break repair, and transformation through multiple interactions with components in infected and transformed cells. Conservation of several cysteine and histidine residues among E4 34k sequences from a variety of adenovirus serotypes suggests the presence of a zinc binding domain important for function. Consistent with the hypothesis that E4 34k is a zinc metalloprotein, zinc binding by baculovirus-expressed E4 34k protein was demonstrated in a zinc blotting assay. To investigate the relationship between the potential zinc-binding region and E4 34k function, a series of mutant genes containing single amino acid substitutions at each of the conserved cysteine and histidine residues in E4 34k were constructed. The mutant proteins were examined for the ability to complement the late protein synthetic defect of an E4 deletion mutant, to physically interact with the viral E1b 55-kDa protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of cysteine and histidine residues required for stimulation of late gene expression, physical interaction with E1b 55k, and p53 destabilization. These data suggest that a zinc-binding domain participates in the formation of the E4 34k-E1b 55k physical complex and that the complex is required in late gene expression and for p53 destabilization.

Subcellular Sorting of Small Membrane-Associated Triple Gene Block Proteins: TGBp3-Assisted Targeting of TGBp2

We studied subcellular distribution of green fluorescent protein (GFP)-tagged movement proteins encoded by the second and the third genes of poa semilatent hordeivirus (PSLV) triple gene block (TGB), 15K TGBp2 and 18K TGBp3. GFP-15K transiently expressed in Nicotiana benthamiana leaf epidermal cells was associated with the endomembrane system elements. GFP-18K appeared in the membrane bodies at cell periphery. Mutation analysis demonstrated that subcellular targeting of GFP-15K depended on the protein transmembrane segment(s), whereas the TGBp3 central hydrophilic region was responsible for targeting of GFP-18K. Coexpression of GFP-15K with the intact 18K protein induced drastic changes in the TGBp2 localization: GFP-15K appeared in the cell peripheral bodies similar to those in the cells expressing GFP-18K alone. Coexpression experiments with mutant forms of both proteins argue against involvement of direct interaction between small TGB proteins in the TGBp3-assisted targeting of TGBp2 to the cell peripheral compartments. This conclusion was further confirmed by similar effects on the PSLV 15K TGBp2 localization induced by TGBp3 proteins of PSLV and potato virus X, which have no detectable sequence similarity to each other.

Identification and initial characterization of elastase activity associated with Vibrio cholerae.

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14-18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus.

Similarities in the genome organization of tobacco rattle virus and pea early-browning virus isolates that are transmitted by the same vector nematode.

Although sequence data have been obtained for several tobravirus isolates, only two of these isolates are nematode-transmissible. Tobacco rattle virus (TRV) PpK20 is transmitted by Paratrichodorus pachydermus, whereas pea early-browning virus (PEBV) TpA56 is transmitted by Trichodorus primitivus. To clarify whether differences in the genome structure of these isolates are relevant to the specificity of interactions with particular vector nematodes, or merely reflect a taxonomic difference between TRV and PEBV, we have sequenced RNA2 of a new isolate of TRV (TpO1) that is transmitted by the same vector nematode as PEBV TpA56 but is not transmitted by the nematode vector of TRV PpK20. TRV TpO1 RNA2 encodes, in 5' to 3' order, a coat protein (CP), a 9K protein, a 2b (29K) protein and a 2c (18K) protein. Amino acid sequence comparison shows that both the CP and 2b proteins of TRV TpO1 resemble more closely the analogous proteins from PEBV TpA56 than those from TRV PpK20. Also, the TRV TpO1 9K protein has similarities with the PEBV 9K protein whereas this protein is lacking in TRV PpK20.

A protein of the basal lamina of the sea urchin embryo.

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.

Apolipoprotein E is Present in Primary Localized Cutaneous Amyloidosis

Apolipoprotein E (apoE) is one of the amyloid associated proteins that is found in the amyloid plaque of Alzheimer's disease and systemic amyloidosis. ApoE might play an important part in the etiology of Alzheimer's disease by functioning as a "pathologic chaperone" to promote the formation of amyloid filaments. In this study, we investigated whether apoE is associated with amyloid deposits of primary localized cutaneous amyloidosis using immunohistochemistry, immunogold electron microscopy, and immunoblotting. The subjects consisted of 12 patients with lichen amyloidosus and one patient with macular amyloidosis. Light microscopically, amyloid deposits in the dermal papillae were round in shape and stained with Congo red. Immunohistochemically, apoE was detected in amyloid deposits in all the cases examined. Immunogold electron microscopy showed apoE immunoreactivity on the amyloid deposition. Immunoblots of amyloid-positive skin showed 35K and 14K proteins, which were taken to be apoE and its fragment, respectively. In normal skin extract, only the 35K protein was detected by the anti-human apoE. Moreover, the intensity of the amyloid-positive skin sample was stronger than that of the normal skin sample. Monoclonal anti-cytokeratin antibody reacted with the 45K protein of the amyloid-positive skin extract. These results indicate that apoE is a component of primary localized cutaneous amyloidosis, and that it might play an important role in primary localized cutaneous amyloidosis.

Comparative Sequence Analysis of Biologically Distinct Isolates of Citrus Tristeza Virus in Japan.

Many Japanese isolates of citrus tristeza virus (CTV) vary in the severity of symptoms which they induce. To study the molecular variability of these isolates, a reverse transcription polymerase chain reaction technique (RT-PCR) was used to amplify part of the CTV genome. PCR products included the 3'-end of the 27K protein gene, most of the 5'-end of the coat protein (CP) gene and the intercistronic region (GAP) between the two genes. Nucleotide sequence analysis of PCR products classified the 13 nucleotide sequences obtained from 12 Japanese isolates of CTV into three groups. Group I contains severe isolates (1215A, 1513A, 1595A-1, HS34, KS3A2), group II mild isolates (BOUQA, M12A, M15A, M27A), and group III severe (S5A2, S5agA) and mild (M23A, 1595A-2) isolates. In the GAP region, a deletion of bases in the Japanese severe CTV isolates may be useful in developing a molecular technique to discriminate CTV isolates. The amino acid residue at position 124 of the coat protein appeared previously to be the most conserved and isolate-specific for mild and severe strains. Among Japanese CTV isolates, however, two mild isolates M15A and M23A had the same residue at position 124 as severe isolates. Based on these results, a greater diversity exists among Japanese CTV isolates than among those in other citrus producing areas.

Nucleotide Sequence and Genomic Organization of Acyrthosiphon Pisum Virus

The nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3′-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a −1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3′-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1- (34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to thePicornaviridae.

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture.

The life span of normal human cells in culture is extended by two to four total life spans following retrovirus-mediated transfer of the adenovirus type 12 E1B 54,000-molecular-weight protein (54K protein). This extension of the in vitro growth potential was accomplished without any of the obvious changes in morphology or growth properties that are usually associated with viral transformation. These 54K+ cells escape the normal senescence checkpoint (M1) and show a very extended secondary growth phase. The 54K+ human cells eventually enter crisis (M2), which does not appear to be due to either telomere attrition or the activation of the senescence-associated proteins p21SdilCipIWaf1 and p16INK4A. Even in the absence of telomerase activity, high-molecular-weight heterogeneous telomeres are produced and maintained in both 54K+ adult dermal fibroblasts and embryo kidney cells, indicating that the 54K protein may interfere with the normal metabolism of telomeric structures during cell division. These findings are discussed with reference to the known ability of the 54K protein to influence p53 function.

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.

Acellular pertussis vaccines containing genetically detoxified pertussis toxin induce long-lasting humoral and cellular responses in adults

New generation pertussis vaccines, containing only purified Bordetella pertussis antigens, have been proven safe, immunogenic and efficacious. They have, however, raised new questions regarding the mechanism of protection from whooping cough and the duration of the immune response following vaccination. In addition to the antibody (Ab) titer, the level of pertussis toxin (PT) neutralizing antibodies may be very important in protection and the role of cell-ediated immunity needs to be defined. We have previously reported the safety and immunogenicity results of two phase I trials in adult volunteers with two acellular pertussis vaccines containing genetically detoxified PT alone or in combination with filamentous hemagglutinin (FHA) and 69K protein. In this work, we present the results of a long term follow-up study of the immune response in the same vac cinees. We evaluated the Ab response, the PT neutrealizing titer and the peripheral blood T cell response up to 4 years following vaccination. Our results show that in adults the level of antibodies to PT, FHA and 69K and the PT neutralizing titers slightly decline between 2.5 and 12 months after the last vaccine dose, but they remain high in the following 2–4 years, showing levels 10–100 times higher than pre-vaccination values. The T cell responses were more heterogeneous among vaccinees but they did not show any significant decline throughout the period monitored.

Identification of actin-binding proteins from sea urchin eggs by F-actin affinity column chromatography.

Novel F-actin binding proteins of sea urchin eggs were searched for in order to study regulation of the actin cytoskeleton during fertilization and cell division. An extract of unfertilized eggs was analyzed by F-actin column chromatography. Several previously characterized F-actin-modulating proteins such as spectrin, myosin, and fascin bound to the column. The eluates from the column also contained proteins having apparent molecular weights of 225K, 150K, 70K, 60K, 45K, 40K, 38K, 36K, 34K, 20K, and 15K, which were thought to be novel cytoskeletal proteins judging from their molecular weights and non-reactivity to antibodies against previously characterized F-actin-modulating proteins. Most of the proteins in the F-actin column eluates co-sedimented with F-actin. Partial amino acid sequences of the peptides derived from the 45K and 40K proteins showed that these proteins are homologous to Arp3 and Arp2 subfamilies of actin-related proteins, respectively. The 150K protein seemed to be an unconventional myosin, that belongs to myosin VI subfamily. Amino acid sequences of two fragments from the 60K protein showed homology to that of coronin. The 150K protein was localized by immunofluorescence microscopy to the cleavage furrows in both whole cell sample and isolated cortex of dividing eggs. The 70K protein was uniformly localized in the cortical layer in the whole egg, but weak staining of the cleavage furrow region with the antiserum was observed in the isolated cortex. The 60K protein was localized to both the bulk cortical layer and the cleavage furrow, but the modes of localization were different.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67,348 polypeptide encoded by genome segment 8.

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSVS8 is 1914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1810 which is capable of encoding a protein of M(r) 67,348. The N-terminal amino acid sequence of a approximately 43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a approximately 43K major capsid protein.

The 24 kDa proteinases of comoviruses are virus-specific in cis as well as in trans.

To investigate the specificity of comoviral 24 kDa ('24K') proteinases, a full-length cDNA copy of red clover mottle virus (RCMV) RNA 1 has been cloned downstream of a T7 promoter. Translation in rabbit reticulocyte lysates of in vitro transcripts from this clone resulted in the synthesis of a 200K protein which was processed in a manner similar to that of the equivalent protein from cowpea mosaic virus (CPMV). Full-length cDNA clones of the RNA 1 molecules of RCMV and CPMV were used to create hybrid RNA 1 molecules. RNA transcribed in vitro from these hybrids was translated in vitro and the ability of the 24K proteinase from one comovirus to cleave the 32K/170K processing site from the other assessed. The results of the experiments show that the 24K proteinases are virus-specific in cis.