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Nucleotide changes in sequential variants of influenza virus hemagglutinin genes and molecular structures of corresponding monoclonal antibodies specific for each variant.

We have generated four monoclonal antibodies specific for one or more members of a series of two sequentially derived PR8 influenza virus variants. Three of these antibodies share cross-reactive idiotypes. The amino acid sequences of these antibodies were determined, and it was found that two of these antibodies use genes from the VH7183 family, whereas the third uses a gene from the VHJ558 family. All four monoclonal antibodies derive from different families of genes encoding the variable region of the kappa chain. The RNA sequence of the parent PR8 virus as well as the RNA sequences of the sequential variants were also determined, and it was demonstrated that the variant hemagglutinin molecules differed from the parent molecule by only a single amino acid interchange. Despite these subtle differences in antigenic structures of hemagglutinin, and the cross-reactive idiotype of the antibodies, their primary structures were very different. These data reinforce the idea that a wide variety of antibody structures exist which are directed against subtly different structures in biologically important antigens.

Amino Acid Sequence↗

The mechanism of isoluminant chromatic motion perception.

An isoluminant chromatic display is a color display in which the component colors have been so carefully equated in luminance that they stimulate only color-sensitive perceptual mechanisms and not luminance-sensitive mechanisms. The nature of the mechanism by which isoluminant chromatic motion is perceived is an important issue because color and motion processing historically have been associated with different neural pathways. Here we show that isoluminant chromatic motion (i) fails a pedestal test, (ii) has a temporal tuning function that declines to half-amplitude at 3-6 Hz, and (iii) is perceived equally well when the entire motion sequence is presented monocularly (entire motion sequence to one eye) versus interocularly (the frames of motion sequence alternate between eyes so that neither eye individually could perceive motion). These three characteristics indicate that chromatic motion is detected by the third-order motion system. Based on this theory, it was possible to take a moving isoluminant red-green grating and, by simply increasing the chromatic contrast of the green component, to generate the full gamut of motion percepts, from compelling smooth motion to motion standstill. The perception of motion standstill when the third-order mechanism is nullified indicates that there is no other motion computation available for purely chromatic motion. It follows that isoluminant chromatic motion is not computed by specialized chromatic motion mechanisms within a color pathway but by the third-order motion system at a brain level where binocular inputs of form, color, depth, and texture are simultaneously available and where selective attention can exert a major influence.

Calibration↗

Electrical inexcitability of the frog neuromuscular synapse.

Frog muscle endplates were explored with an extracellular microelectrode. An intracellular microelectrode nearby simultaneously monitored invasion of the endplate by a spike directly evoked by a third microelectrode placed away from the endplate in the same fiber. External positivities were seen only at sites generating miniature endplate potentials. The external positivity reached a maximum prior to the internally recorded potential and was followed by a small late negativity. Small movements away from active synaptic sites resulted in positive-negative-positive potential sequences characteristic of activity and propagation. Since the external potential is a function of membrane current, the absence of negativity associated with the rising phase of the spike indicates the absence of inward current at synaptic sites. Thus, the synaptic membrane appears not to be excited by a depolarization of the magnitude of an action potential. In an Appendix it is shown that the late negativity and earlier maximum of the external potential can be accounted for by capacitative current through passive membrane.

Action Potentials↗

TE-AFLP: combining rapidity and robustness in DNA fingerprinting.

A new type of fingerprinting technique is presented, based on amplified fragment length polymorphism (AFLP). Rather than two endonucleases as in AFLP, we propose the use of three enzymes, hence the method is called three endonuclease (TE)-AFLP. Genomic DNA is digested and two sets of adapters are selectively ligated onto the restriction fragments in a single reaction volume. No adapters complementary to the ends generated by a frequent cutter are added. Due to the addition of a third endonuclease, the TE-AFLP method provides a high discriminatory power and a reduction in the number of bands. The latter makes it especially suitable for the analysis of complex genomes. TE-AFLP fingerprints are suitable for detection by automatic fluorescent sequencers and are obtained in less than half the time and at reduced costs compared to a typical AFLP. The reliability of this method was investigated by determining the influence of varying digestion, ligation and PCR components on the fingerprint. Moreover, cross-experiments to study inheritance of loci were performed with a primitive insect and with tomato strains. The features of TE-AFLP are discussed in comparison with conventional AFLP.

Animals↗

Location of an immunizing determinant within polypeptide VP1 of type O aphthovirus.

VP1 is the only structural polypeptide of aphthovirus able to stimulate the production of neutralizing antibody. The region of VP1 responsible for this activity was located by testing various proteolytic fragments of VP1 for their ability to compete for virus-specific antibodies in serum raised against the intact polypeptide. No antigenic activity could be detected in VP1 fragments isolated from trypsin-treated virus. Controlled digestion revealed that trypsin cleaved VP1 in four places in a preferred order, whereas chymotrypsin cut at a maximum of two sites. The initial cuts by the two proteases were made very close to each other, and in each case resulted in a greatly reduced affinity of the VP1 fragments for virus-specific antibodies in anti-VP1 serum. In contrast, aphthovirus was resistant to Staphylococcus aureus V8 protease, and treatment of isolated VP1 with this protease generated a peptide of mol. wt. 8500 which competed efficiently with virus for antiserum to VP1 and for an absorbed antiviral serum specific for trypsin-sensitive sites. The results indicate that these antisera interact specifically with aphthovirus at a single antigenic determinant located on VP1 approximately two-thirds of the way along the polypeptide sequence.

Amino Acid Sequence↗

The amino acid sequence of wheat histone H2B(2). A core histone with a novel repetitive N-terminal extension.

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphylococcus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1.

Amino Acid Sequence↗

Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons.

Recently, two members of a new family of Ca2+ channel alpha1 subunits, alpha1G (or CavT.1) and alpha1H (or CavT.2), have been cloned and expressed. These alpha1 subunits generate Ba2+ currents similar to the T-type Ca2+ currents present in sensory neurons. Here, we use three methods to investigate whether the T currents of nodosus ganglion neurons are encoded by members of the CavT family. PCR detected the presence of mRNA encoding both alpha1G and alpha1H, as well as a third highly related sequence, alpha1I. In situ hybridizations performed on nodosus ganglia demonstrate a high expression of alpha1H subunit RNAs. Transfection of nodosus ganglion neurons with a generic antisense oligonucleotide against this new alpha1 subunit family selectively suppresses the low-voltage-activated Ca2+ current. The antisense oligonucleotide effect increased with time after transfection and reached a maximum 3 d after treatment, indicating a 2-3 d turnover for the alpha1 proteins. Taken together, these results suggest that the T-type current present in the sensory neurons is mainly attributable to alpha1H channels. In addition, taking advantage of the high specificity of the antisense ON to the cloned channels, we showed that T-type currents greatly slowed the repolarization occurring during an action potential and were responsible for up to 51% of the Ca2+ entry during spikes. Therefore, the antisense strategy clearly demonstrates the role of low-voltage-activated Ca2+ current in affecting the afterpotential properties and influencing the cell excitability. Such tools should be beneficial to further studies investigating physiological roles of T-type Ca2+ currents.

Action Potentials↗

Limited proteolysis and active-site studies of the first multienzyme component of the erythromycin-producing polyketide synthase.

The domain structure of the 6-deoxyerythronolide B synthase 1 component of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea has been investigated using limited proteolysis and active-site labeling. Trypsin, elastase, endoproteinase Glu-C, and endoproteinase Arg-C were used to cleave the multienzyme, and the sizes of the resulting fragments were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The location of fragments within the primary structure was established by N-terminal sequence analysis. The cleavage pattern followed domain boundaries previously predicted on the basis of sequence alignments, but many predicted interdomain regions were not cleaved, even under the harshest conditions used. Initial proteolysis generated three large fragments: an N-terminal fragment (about 60 kDa) housing an acyltransferase-acyl carrier protein di-domain; a central fragment (about 90 kDa) containing a ketosynthase-acyltransferase di-domain; and a C-terminal fragment (about 220 kDa) containing the remaining six domains of the multienzyme, including the third acyltransferase. The intact multienzyme behaves as a dimer of molecular mass 660 kDa on gel filtration; and the C-terminal fragment remains dimeric. However, the N-terminal and central fragments appear to be monomeric species. After proteolysis of the multienzyme, the N-terminal di-domain was found to be specifically labeled after incubation with [14C]propionyl-CoA, providing the first evidence for its proposed role as a "loading domain" for the propionate starter unit. In contrast, the other two fragments were specifically acylated by [14C]methylmalonyl-CoA, indicating that both the other two acyltransferases remain enzymatically active after proteolysis.

Amino Acid Sequence↗

G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin.

Amino Acid Sequence↗

A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop.

The mouse D3 dopamine receptor has been cloned from olfactory tubercle cDNA using polymerase chain reaction and has been found to exist in two alternatively spliced forms. These two mRNA isoforms differ by the presence or absence of 63 base pairs (bp), which encode 21 amino acids in the putative third cytoplasmic loop of the receptor. The longer form corresponds to the previously reported rat D3 dopamine receptor, to which it bears sequence homology of 94%. Northern blot analysis shows the mouse D3 receptor to be most abundant in the olfactory tubercle. Expression studies show the novel short D3 isoform to bind dopaminergic ligands with a D3-like pharmacological profile. Polymerase chain reaction analysis on different mouse brain regions shows the long and short D3 receptors to be present in the same tissues, the longer form invariably being the predominant one. Analysis of the gene for the mouse D3 dopamine receptor shows that no separate exon encodes the 63-bp stretch and reveals the presence of a consensus sequence for an acceptor site at the 3' end of the 63-bp stretch. This suggests that an internal acceptor site in the exon coding for the distal part of the third cytoplasmic loop directs alternative splicing of the D3 dopamine receptor.

Alternative Splicing↗

Protein interactions with DNA elements in variant equine infectious anemia virus enhancers and their impact on transcriptional activity.

The long terminal repeats (LTRs) from various cloned equine infectious anemia virus (EIAV) proviruses differ significantly, but all contain cis-acting DNA elements identical to MDBP-, PEA2-, AP-1-, and PU.1 (ets)-binding sites. A prototype EIAV LTR would contain one of each of these conserved elements. The LTR variations originate from the insertion of novel sequences between the PEA2 and AP-1 elements in the transcriptional enhancer unit. Viewed in this way, the LTR from provirus clone lambda 12 has an 11-bp insertion containing a PEA2 site and the LTR of the lambda 6 provirus has a 31-bp insertion/duplication containing PEA2, AP-1, and PU.1 sites. Two other LTRs were cloned by amplification of cDNAs from the persistently infected cell line, EIAV-FEA. A third LTR was generated by site-directed mutagenesis of one of the LTRs from EIAV-FEA cells. The latter three had a single base change in the element next to the TATA box that abolished PU.1 binding; however, the variable regions of these LTRs were shown by gel mobility shift assays to contain one or two PU.1 sites. One variable region was shown to have an octamer site overlapping its tandem PU.1 elements. Basal, PMA-activated, and Tat trans-activated transcriptional activities of the LTRs were compared in several different cell lines by transient transfection. The various promoters displayed different relative levels of activity depending on the cell line used and the condition of activation. This natural set of variant promoters may help define how changes in the components of the transcription complex influence transactivation by Tat. The diverse LTRs could endow their respective proviruses with a unique pattern of expression and activation in vivo.

Base Sequence↗

The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli.

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT-con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT-con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.

Adaptation, Physiological↗

A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease.

Proteins can be remarkably tolerant of major mutational changes. Sites that accomodate large insertions without loss of function ("permissive" sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding. The identification of such sites can aid in the engineering of functional derivatives of a protein with novel properties. To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertions in cloned genes. In a first step, a beta-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon derivative into the target gene. Requiring beta-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the transposon relative to the target gene. In a second step, most of the transposon sequences are excised in vitro, leaving the in-frame insertion. Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety of proteins. As a test case, a set of 31-codon insertions in the Escherichia coli lac permease gene was generated. The lactose transport activities of the mutant proteins followed a simple pattern: most of the proteins (10/12) with insertions in sequences thought to face the cytoplasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive. The only exceptions were two inactive proteins with insertions in the third cytoplasmic region. Most of the inactive proteins were detected at reduced levels in cells, presumably due to proteolytic breakdown. These studies thus illustrate the use of the new method to identify permissive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease. In addition to screening for permissive sites, 31-codon insertion mutagenesis may be useful in epitope-tagging proteins at multiple internal positions, in analyzing membrane protein topology, and in dissecting structure-function relationships in proteins.

Amino Acid Sequence↗

Chemical interactions between amino acid and RNA: multiplicity of the levels of specificity explains origin of the genetic code.

The emergence of the genetic code remains an enigma. Proposed mechanisms are based on random, historical, thermodynamic and natural selection. However, they introduce chance as a key factor for overcoming the difficulties encountered by the model. We propose here a model in which three successive levels of chemical specificity generated the nucleotide assignments of amino acids in the genetic code. The first level results from hydrophobic and stereospecific interactions between amino acids and short oligonucleotides (termed oligons). The second and third levels of specificity are determined by conditions of energy transfer from loaded oligons (amino acid-oligomer covalently linked) to formation of phosphodiester bond (second level of specificity) and peptidic bond (third level of specificity), while these reactions are catalyzed by RNA templates. This model is sustained by the relationships observed between dipole moments of the nucleotides (forming the anticodon) and reactivity of the amino acyl linkage of the loaded oligon. Moreover, analysis of modern tRNAs reveals that they were probably generated by loose duplication of the nucleotide sequence forming the oligons, after emergence of the 'genetic code.' Indeed, the similarity of nucleotide composition with that of the anticodon decreases with the tRNA domain's distance from the anticodon, but the acceptor stem is relatively more similar to the anticodon than other stems closer to it. This would be because energy transfer constraints that existed between anticodon and amino acid in prebiotic loaded oligonucleotides still affect the structures of modern tRNA acceptor stems. In the model presented, the genetic code is inherent to the most archaic 'molecular physiology' in protolife, even before emergence of a functional 'protein world.' Simple physical processes, in which a level of specificity is integrated in an emerging meta-structure expressing new properties, generate a parsimonious and realistic explanation of emergence of the genetic code.

Amino Acids↗

The role of the genetic code in generating new coding sequences inside existing genes.

The genetic code has a very interesting property--it generates an open reading frame (ORF) inside a coding sequence, in a specific phase of the antisense strand with much higher probability than in the random DNA sequences. Furthermore, these antisense ORFs (A-ORFs) possess the same features as real genes--the asymmetry in the nucleotide composition at the first and second positions in codons. About two thirds of the 2997 overlapping ORFs in the yeast genome possess this feature. Thus, the question arises: has this feature of the genetic code been exploited in the evolution of genes? We have searched the FASTA data bases for homologies with the antisense translation products of a specific class of genes and we have found some sequences with relatively high homology. Many of them have scores which could be randomly found in the searched data bases with a probability lower than 10(-6). We conclude that some genes could arise by positioning a copy of the original gene under a promoter in the opposite direction in such a way that both, the original gene and its copy initially use the same nucleotides in the third, degenerated positions in codons.

DNA, Antisense↗

Chimeric dopamine-norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium.

The dopamine (DA) and norephinephrine (NE) transporters demonstrate important differences in their selectivity for catecholamines and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+), yet their primary sequences and predicted topology are strikingly similar. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between the DA and NE transporters, a series of recombinant chimeras was generated by a restriction site-independent method and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE, and MPP+. These studies also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared with NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These structure-function studies using chimeras of members of the superfamily of Na(+)- and Cl(-)-dependent transporters provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.

1-Methyl-4-phenylpyridinium↗

Isolation of single stranded DNA related to the transcriptional activity of animal cells.

Single stranded DNA (s.s.DNA) comprising 1-2% of the total nuclear DNA was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA3 of embryonic chick cells, labeled for several cell generations with 3H-thymidine. Small quantities of 3H-DNA were annealed with a large excess of unlabeled DNA or polysomal RNA from chick embryos. Hybridization kinetics (monitored by the use of SI nuclease digestion, hydroxyapatite chromatography and thermalfusion), indicated that s.s.DNA belongs to the non repetitious fraction of the cell genome. One third represents DNA sequences engaged in the transcription of messenger RNA's.

Animals↗

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Animals↗