The role of stigma in reasons for HIV disclosure and non-disclosure to children.

This study examined how stigma may impact HIV-positive women's disclosure to their children. Participants included HIV-infected women recruited from AIDS service organizations located in large midwestern cities. Using a questionnaire and guided interview, quantitative data were obtained regarding perceived HIV-related stigma and rates of maternal disclosure to children aged 5-18. According to the Kruskal-Wallis test, comparison between the disclosure groups showed non-significant differences in the total score of HIV stigma (chi(2)=0.518 with df = 2, p=0.77).

Effect of local sequence inversions on the crystalline antiparallel beta-sheet lamellar structures of periodic polypeptides: implications for chain-folding.

The crystal structure and texture of the monodisperse periodic polypeptide [(AG)3EG(GA)3EG]10 (poly(+/-AG)3EG: A=alanine, G=glycine, E=glutamic acid) were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy. Structure determination was aided by comparison with the recently described structure for the related periodic polypeptide [(AG)3EG]36 by Krejchi et al. (Macromolecules 1997;30:5012). Texture-oriented samples of poly(+/-AG)3EG were obtained by crystallization of the polymer from aqueous formic acid solution. The evidence supports an antiparallel (ap) beta-sheet protein structure and the X-ray diffraction signals index on an orthorhombic unit cell with parameters: a=0.950 nm (hydrogen-bond direction), b=1.052 nm (apbeta-sheet stacking direction), c=6.95 nm (chain direction). The absence of the (010) diffraction signal, a prominent signal in the poly(AG)3EG diffraction pattern, implies that the apbeta-sheets are 'apolar', i.e. both surfaces are equally populated with alanyl methyl groups. Selective line broadening of wide-angle diffraction signals with l not equal to 0 gives an estimated crystal size of approximately/= 4 nm in the chain direction. This observation, coupled with the appearance of low-angle particle interference peaks, indicates a crystal thickness considerably less than the chain length and suggests an adjacent-re-entry chain-folded lamellar structure incorporating the apbeta-sheet architecture. The polypeptide folds through gamma-turns, in-phase with the pseudo-octapeptide repeat; the glutamic acid residues occur on the lamellar surfaces. These results and those from the crystalline lamellae of poly(AG)3EG suggest that beta-turns are not compatible with these repetitively stacked apbeta-sheet structures. This implies that intersheet interactions of alanyl methyl groups and glycyl alpha-protons are not sufficiently strong to dictate the folding geometry in these structures.

Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.

A critique of the usefulness of inferential statistics in applied behavior analysis.

Researchers continue to recommend that applied behavior analysts use inferential statistics in making decisions about effects of independent variables on dependent variables. In many other approaches to behavioral science, inferential statistics are the primary means for deciding the importance of effects. Several possible uses of inferential statistics are considered. Rather than being an objective means for making decisions about effects, as is often claimed, inferential statistics are shown to be subjective. It is argued that the use of inferential statistics adds nothing to the complex and admittedly subjective nonstatistical methods that are often employed in applied behavior analysis. Attacks on inferential statistics that are being made, perhaps with increasing frequency, by those who are not behavior analysts, are discussed. These attackers are calling for banning the use of inferential statistics in research publications and commonly recommend that behavioral scientists should switch to using statistics aimed at interval estimation or the method of confidence intervals. Interval estimation is shown to be contrary to the fundamental assumption of behavior analysis that only individuals behave. It is recommended that authors who wish to publish the results of inferential statistics be asked to justify them as a means for helping us to identify any ways in which they may be useful.

Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length.

Solutions and melts of stiff ('rod-like') macromolecules often exhibit nematic liquid crystalline phases characterized by orientational, but not positional, molecular order. Smectic phases, in which macromolecular rods are organized into layers roughly perpendicular to the direction of molecular orientation, are rare, owing at least in part to the polydisperse nature (distribution of chain lengths) of polymers prepared by conventional polymerization processes. Bacterial methods for polypeptide synthesis, in which artificial genes encoding the polymer are expressed in bacterial vectors, offer the opportunity to make macromolecules with very well defined chain lengths. Here we show that a monodisperse derivative of poly(gamma-benzyl alpha,L-glutamate) prepared in this way shows smectic ordering in solution and in films. This result suggests that methods for preparing monodisperse polymers might provide access to new smectic phases with layer spacings that are susceptible to precise control on the scale of tens of nanometres.

Effects of amino acid side-chain volume on chain packing in genetically engineered periodic polypeptides.

The fidelity of bacterial protein synthesis allows the production of architecturally well-defined polymeric materials through precise control of chain length, sequence, stereochemistry, and interchain interactions. In the present paper, we examine the relation between amino acid residue volume and crystalline unit cell dimensions, in a set of periodic protein polymers of repeating unit sequence -(AlaGly)3-X-Gly-, where X is Asn, Phe, Ser, Val, or Tyr. The proteins were overexpressed in Escherichia coli, purified by simple procedures based on acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate, and processed to form oriented crystalline mats by precipitation from formic acid under mechanical shear. X-ray diffraction analyses revealed that the basic structures of the -(AlaGly)3-X-Gly- polymers are identical to that previously reported for [(AlaGly)3-GluGly]36, [Krejchi, M.T., Atkins, E.D.T., Waddon, A.J., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1994) Science 265, 1427-1432], with the oligoalanylglycine segments forming antiparallel beta-sheets and the substituted amino acids occurring within three-residue folds at the lamellar surfaces. The X-ray diffraction signals for each member of the family index on an orthorhombic unit cell; the a-axis (hydrogen bond direction) and c-axis (chain direction) spacings remain invariant but the b-axis (sheet stacking direction) spacing increases with increasing volume of the substituted amino acid. The results obtained from a variant with alternating Glu and Lys substitution at the X position, together with the results previously reported for poly(L-alanylglycine) [Panitch, A., Matsuki, K., Cantor, E.J., Cooper, S.J., Atkins, E.D.T., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1997) Macromolecules 30, 42-49] are included for comparison. The average intersheet stacking distance (b/2) increases linearly with the volume of the amino acid inserted at position X. Because the chain-folded lamellar architecture adopted by these periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity, these results illustrate a new approach to the engineering of intermolecular interactions in polymeric solids.

Functional analysis of ribosomal protein L2 in yeast mitochondria.

The yeast nuclear gene RML2, identified through genomic sequencing of Saccharomyces cerevisiae chromosome V, was shown to encode a mitochondrial homologue of the bacterial ribosomal protein L2. Immunoblot analysis showed that the mature Rml2p is a 37-kDa polypeptide component of the mitochondrial 54 S large ribosomal subunit. Null mutants of RML2 are respiration-deficient and convert to [rho-] or [rho degrees ] cytoplasmic petites, indicating that Rml2p is essential for mitochondrial translation. RML2 is regulated transcriptionally in response to carbon source and the accumulation of Rml2p is dependent on the presence of the 21 S large rRNA. Site-directed mutagenesis showed that a highly conserved 7-amino acid sequence (Val336 to Asp342) of Rml2p is essential for function. Substitution of Gln for His-343, the most highly conserved histidine in the L2 protein family, caused cold-sensitive respiratory growth but did not affect the assembly of 54 S ribosomal subunits. Mitochondrial protein synthesis was normal in the His343 to Gln (H343Q) mutant grown at the permissive temperature (30 degrees C) and was severely impaired after growth at the nonpermissive temperature (18 degrees C). His343 corresponds to His229 in Escherichia coli L2, which has been implicated in a direct involvement in peptidyl transferase activity. The conditional phenotype of the H343Q mutant indicates that His343 is not essential for peptidyl transferase activity in yeast mitochondria.

Molecular genetics of the peptidyl transferase center and the unusual Var1 protein in yeast mitochondrial ribosomes.

Mitochondria possess their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. In yeast, Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Var1, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus. The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria. Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Var1 protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.

Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus.

The Var1 protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Var1p, we have engineered yeast strains for the expression of this protein from a nuclear gene, VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria. VAR1U was tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts '3-end processing of the VARI mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carrying VAR1U under ADH1 promoter control, thus proving that VAR1 function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation of VAR1 from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure-function relationships in the unusual Var1 protein.

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.

Implications of a functional large ribosomal RNA with only three modified nucleotides.

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.

The role of nucleotide modifications in the yeast mitochondrial ribosome.

Post-transcriptionally modified nucleotides in ribosomal RNA, and the modifying enzymes themselves, could influence the assembly, structure, and function of the ribosome. The PET56 gene in yeast encodes the enzyme responsible for formation of 2'-O-methylguanosine at a specific nucleotide in the peptidyl transferase center of the mitochondrial large subunit ribosomal RNA. While PET56 is normally essential for the formation of functional mitochondrial ribosomes, extragenic mutations have been obtained that suppress, albeit weakly, pet56 loss-of-function mutations. Thus neither the Pet56p-catalyzed ribose methylation nor the Pet56 protein itself is absolutely required for the synthesis of a functional ribosome.

Chemical sequence control of beta-sheet assembly in macromolecular crystals of periodic polypeptides.

A family of uniform periodic polypeptides has been prepared by bacterial expression of the corresponding artificial genes, with the objective of exploring the potential for control of supramolecular organization in genetically engineered protein-based polymeric materials. The repeating units of the polypeptides consist of oligomeric alanyl-glycine sequences interspersed with glutamic acid residues inserted at intervals of 8 to 14 amino acids. Crystallization of such materials from formic acid produces beta-sheet structures in the solid state, as shown by vibrational spectroscopy, nuclear magnetic resonance spectroscopy, and wide-angle x-ray diffraction. The diffraction results, together with observations from electron microscopy, are consistent with the formation of needle-shaped lamellar crystals whose thickness is controlled by the periodicity of the primary sequence. These results can be used to control solid-state structure in macromolecular materials.

T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.

Functional requirement of a site-specific ribose methylation in ribosomal RNA.

The product of the PET56 nuclear gene of Saccharomyces cerevisiae was shown to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S rRNA). Cells reduced in this activity were deficient in formation of functional large subunits of the mitochondrial ribosome. The purified Pet56 protein catalyzed the site-specific formation of 2'-O-methylguanosine on in vitro transcripts of both mitochondrial 21S rRNA and Escherichia coli 23S rRNA. These results provide evidence for an essential modified nucleotide in rRNA.

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.

HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.

Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection.

Four recombinant vaccinia viruses were engineered for expression of different portions of the Japanese encephalitis virus (JEV) open reading frame. All four recombinant vaccinias contained the NS1 and NS2A genes, and each of these viruses specified the synthesis, glycosylation, and secretion of the nonstructural glycoprotein (NS1). All four recombinants also contained the E gene, and each virus correctly directed the synthesis and glycosylation of the envelope glycoprotein (E). Interestingly, two of these viruses (vP555 and vP650), which expressed the prM gene in addition to E and NS1, produced an extracellular hemagglutinin containing M and E that migrated in sucrose gradients similarly to the slowly-sedimenting hemagglutinin found in the culture fluid of JEV-infected cells. Immunization of 3-week-old mice with the recombinant viruses vP555 and vP658 resulted in immune responses to NS1, whereas only the virus that directed the synthesis of extracellular forms of E (vP555) induced an immune response to E. Both viruses provided protection against lethal challenge with JEV. Animals given two inoculations with vP555 were fully protected from greater than 10,000 LD50 of JEV. This high level of protection was correlated with the production of high titers of neutralizing and hemagglutination-inhibiting antibodies.

The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli.

The antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DEN1) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli. Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs). Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge. MAbs also reacted with several determinants clustered near the N terminus of the NS1 protein (aa 57 to 126). Recombinant fusion proteins containing E. coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice. The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.