The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.     

Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different herpesvirus. Our data show that in gII- PrV, gI(BHV-1) in vitro fully complements the lethal defect associated with lack of gII(PrV). These results conclusively demonstrate that gI(BHV-1) in a PrV background can execute all essential functions normally provided by gII(PrV). They also indicate that the origin of gB-homologous glycoproteins influences the penetration kinetics of herpesviruses.     

The nucleotide sequence of herpes simplex virus type 2 (333) glycoprotein gB2 and analysis of predicted antigenic sites

The gene coding for glycoprotein B2 (gB2) of herpes simplex virus type 2 (HSV-2) strain 333 was mapped and its nucleotide sequence determined. Open reading frame analysis deduced a polypeptide consisting of 902 amino acids and having close homology to gB1 of HSV type 1. Several predicted features of gB2 are consistent with a membrane-bound glycoprotein, i.e., a signal peptide sequence, a hydrophilic extracellular domain containing possible N-linked glycosylation sites, a hydrophobic membrane spanning sequence, and a cytoplasmic domain. Computer analysis on hydrophilicity, accessibility, and flexibility of the gB2 amino acid sequence, produced a composite surface value plot. At least nine major antigenic regions were predicted on the extracellular domain. The amino acids between residues 59-74, 127-139, 199-205, 460-476, and 580-594 exhibited the highest surface values. Comparison of the primary sequence with gB1 revealed localized regions showing amino acid diversity. Several of these locations correspond to major antigenic regions. Chou and Fasman analyses indicated that the amino acid substitutions, between positions 57-66, 461-472, and 473-481, induced changes in the secondary structure of gB. These sites could represent site-specific epitopes in the gB polypeptide.     

Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is integrated into the PrV envelope and can functionally replace glycoprotein gII of PrV.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

Engineering glycoprotein B of bovine herpesvirus 1 to function as transporter for secreted proteins: a new protein expression approach

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. Konig, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-alpha) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-alpha, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-alpha was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-alpha, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-alpha fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems.     

Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product.

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.     

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.     

Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.     

Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO.

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.     

Structure and expression of the herpes simplex virus type 2 glycoprotein gB gene.

The gene for glycoprotein gB2 of herpes simplex virus type 2 strain 333 was cloned, sequenced, and expressed in mammalian cells. The gB2 protein had an overall nucleotide and amino acid sequence homology of 86% with the cognate gB1 protein. However, of the 125 amino acid substitutions or deletions, only 12.5% were conservative replacements. These differences were clustered within an NH2-terminal region, a central region, and a COOH-terminal region, resulting in domains of near identity broken by small regions of marked divergence. Regions of greatest homology included a 90-amino-acid stretch starting at residue 484 and 39 amino acids spanning residues 835 to 873, which cover a rate-of-entry locus mapped to Ala-552 and a syn locus mapped to Arg-857, respectively, in gB1 by Bzik et al. (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984). Pellett et al. (P. E. Pellett, K. G. Kousoulas, L. Pereira, and B. Roizman, J. Virol. 53:243-253, 1985) mapped the mutations in three monoclonal antibody-resistant gB1 mutants between amino acids 273 and 443. These epitopes are included in a region of 98 residues identical between gB1 and gB2. The identity of this protein was verified by placing a truncated gene lacking the 303 carboxyl-terminal amino acids of gB2 into mammalian COS and CHO cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation. This protein will be purified from the stable CHO cell lines and compared with gB1 for immunogenicity and protective efficacy in animal challenge models.     

The bovine herpesvirus 1 maturational proteinase and scaffold proteins can substitute for the homologous herpes simplex virus type 1 proteins in the formation of hybrid type B capsids.

We determined the nucleotide sequence of a 3.5-kb region of the bovine herpesvirus 1 (BHV-1) genome which contained the complete BHV-1 homologs of the herpes simplex virus type 1 (HSV-1) UL26 and UL26.5 genes. In HSV-1, the UL26 and UL26.5 open reading frames encode scaffold proteins upon which viral capsids are assembled. The UL26-encoded protein is also a proteinase and specifically cleaves both itself and the UL26.5-encoded protein. The overall BHV-1-encoded amino acid sequence showed only 41% identity to the HSV-1 sequences and was most divergent in the regions defined to be involved in the scaffolding function. We substituted the proteins encoded by the BHV-1 homologs of the UL26 and UL26.5 open reading frames, expressed in baculovirus, for the corresponding HSV-1 proteins in an in vitro HSV-1 capsid assembly system. The proteins expressed from the BHV-1 UL26 and UL26.5 homologs facilitated the formation of hybrid type B capsids indistinguishable from those formed entirely with HSV-1-encoded proteins.     

Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine cells.

The gene encoding bovine herpesvirus 1 (BHV-1) glycoprotein gIV was mapped, cloned, and sequenced. The gene is situated between map units 0.892 and 0.902 and encodes a predicted protein of 417 amino acids with a signal sequence cleavage site between amino acids 18 and 19. Comparison of the BHV-1 amino acid sequence with the homologous glycoproteins of other alphaherpesviruses, including herpes simplex virus type 1 glycoprotein gD, revealed significant homology in the amino-terminal half of the molecules, including six invariant cysteine residues. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). Constitutive expression of gIV proved toxic to cells, since stable cell lines could only be established when the gIV gene was placed under the control of an inducible promoter. Expression of gIV was cell associated and localized predominantly in the perinuclear region, although nuclear and plasma membrane staining was also observed. Radioimmunoprecipitation revealed that the recombinant glycoprotein was efficiently processed and had a molecular weight similar to that of the native form of gIV expressed in BHV-1-infected bovine cells. Recombinant gIV produced in the transfected bovine cells induced cell fusion, polykaryon formation, and nuclear fusion. In addition, expression of gIV interfered with BHV-1 replication in the transfected bovine cells.     

Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.     

Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants.

In this paper we report the nucleotide sequence and predicted amino acid sequence of glycoprotein B of herpes simplex virus 1 strain F and the amino acid substitutions in the domains of the glycoprotein B gene of three mutants selected for resistance to monoclonal antibody H126-5 or H233 but not to both. Analyses of the amino acid sequence with respect to hydropathicity and secondary structure yielded a two-dimensional model of the protein. The model predicts an N-terminal, 29-amino-acid cleavable signal sequence, a 696-amino-acid hydrophilic surface domain containing six potential sites for N-linked glycosylation, a 69-amino-acid hydrophobic domain containing three segments traversing the membrane, and a charged 109-amino-acid domain projecting into the cytoplasm and previously shown to marker rescue glycoprotein B syn mutations. The nucleotide sequence of the mutant glycoprotein B DNA fragments previously shown to marker transfer or rescue the mutations revealed that the amino acid substitutions cluster in the hydrophilic surface domain between amino acids 273 and 305. Analyses of the secondary structure of these regions, coupled with the experimentally derived observation that the H126-5- and H233-antibody cognitive sites do not overlap, indicate the approximate locations of the epitopes of these neutralizing, surface-reacting, and immune-precipitating monoclonal antibodies. The predicted perturbations in the secondary structure introduced by the amino acid substitutions correlate with the extent of loss of reactivity with monoclonal antibodies in various immunoassays.     

Larval salivary gland secretion proteins in Drosophila structural analysis of the Sgs-5 gene

The structure of the Drosophila melanogaster salivary gland secretion gene Sgs-5 has been determined by DNA sequence analysis of cloned genomic DNA. This developmentally and tissue-specific gene is a member of the third instar intermolt gene set and is under control of the insect molting hormone ecdysterone. RNA protection experiments show that the RNA coding region of Sgs-5 contains 769 nucleotides and is divided into three exons by two small introns. The protein-coding region appears to begin after a short untranslated RNA leader (33 nucleotides) and to result in a protein of 163 amino acids. The first 18 amino acids give the amino-terminal end the highly hydrophobic nature characteristic of a signal peptide.     

Characterization of a cDNA clone for the nonspecific cross-reacting antigen (NCA) and a comparison of NCA and carcinoembryonic antigen

NCA (nonspecific cross-reacting antigen), a glycoprotein found in normal lung and spleen, is immunologically related to carcinoembryonic antigen (CEA), which is found in over 95% of colon adenocarcinomas. From a human genomic library, we previously cloned part of an NCA gene and showed that the amino-terminal region has extensive sequence homology to CEA (Thompson, J. A., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, Ch. W., Riggs, A. D., and Shively, J.E. (1987) Proc. Natl. Acad. Sci. U. S.A. 84, 2965-2969). We now present the nucleotide sequence of a cDNA clone, containing the entire coding region of NCA (clone 9). The clone was obtained from a lambda gt 10 library made from the colon carcinoma cell line SW 403; the clone contains a 34-amino acid leader sequence, 310 amino acids for the mature protein, and 1.4 kilobases of 3'-untranslated region of the NCA gene. A comparison of the NCA sequence to the CEA sequence (Oikawa, S., Nakazato, H., and Kosaki, G. (1987) Biochem. Biophys. Res. Commun. 142, 511-518; Zimmerman, W., Ortlieb, B., Friedrich, R., and von Kleist, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2690-2694) shows that both proteins contain doublets of an immunoglobulin-like domain, of which there are one copy in NCA and three copies in CEA, a 108-amino acid amino-terminal domain with no cysteine residues, and a carboxyl-terminal hydrophobic domain of sufficient length to anchor the glycoproteins in the cell membrane. Overall, the corresponding coding regions possess 85% sequence homology at the amino acid level and 90% homology at the nucleotide level. Forty nucleotides 3' of their stop codons, the CEA and NCA cDNAs become dissimilar. The 108-amino acid amino-terminal region together with part of the leader peptide sequence corresponds exactly to a single exon described in our previous work. The data presented here further demonstrate the likelihood that CEA recently evolved from NCA by gene duplication, including two duplications of the immunoglobulin-like domain doublet of NCA.