Common mechanism controlling phase and antigenic variation in pathogenic neisseriae

The expression of the Neisseria gonorrhoeae opacity protein (Op, protein II), a major antigenic determinant of the outer membrane, is subject to frequent phase transitions. At least nine expression loci (opaE) are involved in the production of a large number of serologically distinct Op types. Using opa-specific oligonucleotides as probes in genomic blots, we detect Op-related gene sequences (opr) in N. meningitidis as well as in N. lactamica. DNA sequence analysis of such opr genes derived from N. meningitidis reveals distinct regions of homology with gonococcal opa E genes. As shown in the immunoblot, the proteins encoded by opa and opr are serologically related. Like the opaE genes, the 5'-coding sequences of the opr genes include a repetitive sequence composed of pentameric CTCTT units. The number of these coding repeat (CR) units is variable. This finding, together with the observation that all opr genes are constitutively transcribed, regardless of the status of protein production, suggests a translational control mechanism identical to that of the opa genes in gonococci. The related structures and control mechanisms of opa and opr genes imply a general significance of their gene products for the pathogenic character of the investigated Neisseria species.     

The control mechanism of opacity protein expression in the pathogenic Neisseriae

The expression of Neisseria gonorrhoeae opacity protein shows frequent phase transitions. The genome contains at least twelve copies of the opa gene. Each copy is complete and different from most of the others in certain hypervariable regions. All opa genes are constitutively transcribed. Part of the leader peptide of all Op's is encoded by repetitive CTTCT pentameric units, the so-called coding repeat (CR). The number of repeat units found in the genes and mRNAs is subject to frequent and precise changes. Such changes affect the expression of individual opa genes at the translational level. This control mechanism is common also to the class 5 proteins of N. meningitidis and the Op-related proteins of N. lactamica.     

Common mechanism controlling phase and antigenic variation in pathogenic Neisseriae

The expression of the Neisseria gonorrhoeae opacity protein (Op, protein II), a major antigenic determinant of the outer membrane, is subject to frequent phase transitions. At least nine expression loci (opaE) are involved in the production of a large number of serologically distinct Op types. Using opa-specific oligonucleotides as probes in genomic blots, we detect Op-related gene sequences (opr) in N. meninglitidis as well as in N. lactamica. DNA sequence analysis of such opr genes derived from N. meninglitidis reveals distinct regions of homology with gonococcal opa E genes. As shown in the immunoblot, the proteins encoded by ops and opr are serologically related. Like the opa E genes, the 5′-coding sequences of the opr genes include a repetitive sequence composed of pentameric CTCTT units. The number of these coding repeat (CR) units is variable. This finding, together with the observation that all opr genes are constitutively transcribed, regardless of the status of protein production, suggests a translational control mechanism identical to that of the opa genes in gonococci. The related structures and control mechanisms of opa and opr genes imply a general significance of their gene products for the pathogenic character of the investigated Neisseria species.     

The repertoire of silent pilus genes in Neisseria gonorrhoeae: evidence for gene conversion

To investigate the significance of silent gene loci for pilus antigenic variation in N. gonorrhoeae, we determined the nucleotide sequence of the major silent locus, pilS1. The pilS1 locus contains six tandem pilus gene copies linked by a 39 bp repeat sequence also present in the expression loci. All silent copies lack the common N-terminal coding sequence of pilin, containing instead variant sequence information that constitutes a semivariable (SV) and a hypervariable (HV) domain. The SV and HV domains are interspersed with short, strictly conserved (C) regions flanking small cassettes of variable sequence information. It appears that such minicassettes from silent copies can be duplicated and transferred to other silent or expression genes by means of gene conversion.     

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.     

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): mapping of homologies in coding and spacer DNA

The linear arrangement and lengths of the spacers and coding regions in the two nonallelic histone gene variant clusters of L. pictus are remarkably homologous by R loop analysis and are similar in general topography to the histone gene repeat units of other sea urchins examined to date. No interventing sequences were detected. The coding regions of these two histone gene variants share considerable sequence homology; however, there are areas of nonhomology in every spacer region and the lengths of the nonhomologous spacers between the H2A and H1 genes are not the same for the two repeat unit classes (inter-gene heterogeneity). Combining length measurements obtained with both R loops and heteroduplexes suggests that the DNA sequences of the analogous leader regions for the two H1 mRNAs are nonhomologous. Similar observations were made for the H4 leader sequences, as well as the trailer region on H2B. S. purpuratus spacer DNA segments share little sequence homology with L. pictus; however, the analgous coding (and possibly flanking) regions have conserved their sequences. The various coding and spacer regions within a repeat unit do not share DNA sequences. Thus certain areas in the sea urchin histone gene repeat units have been highly conserved during evolution, while other areas have been allowed to undergo considerable sequence change not only between species but within a species.     

Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.     

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: Coding region hypervariability and downstream recombinogenic repeats

The surface of the parasitic protozoanTrypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome ofT. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 by downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy. Correspondence to: J.C. Boothroyd     

Rapid and concerted evolution of repeat units in a balbiani ring gene

The Balbiani ring (BR) genes in the midge Chironomus, a genus belonging to Diptera, code for large secretory proteins, used to construct the larval tube. The 15-23-kb long core block in each gene consists of an array of tandemly arranged approximately 200-bp long repeat units, where a single repeat unit is composed of a constant and a subrepeat region. In order to investigate the evolutionary fate of highly repetitive coding DNA, the BR1γ core block in Chironomus pallidivittatus was characterized and compared to the orthologous core block in the sibling species Chironomus tentans. We find that the 75-100 repeat units in the BR1γ core block have evolved in an unusual fashion. In all repeat units the constant regions display an expected high degree of homology between the two species, 94% at the nucleotide level. In contrast, the subrepeat regions in all repeat units have diverged concertedly, both as to length, number and sequence of the subrepeats. The observed changes in all repeat units of the core block probably have occurred after speciation of C. pallidivittatus and C. tentans. These findings demonstrate that a tandemly reiterated coding sequence can rapidly and concertedly convert into a related sequence, much in the same way as has been described for satellite DNA.     

The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features

Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395-5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5' linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.     

A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element

In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences. They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome. These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat. In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.     

Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.     

5S ribosomal RNA genes in six species of Mediterranean grey mullets: genomic organization and phylogenetic inference.

This paper describes a study of the 5S ribosomal RNA genes (5S rDNA) in a group of 6 species belonging to 4 genera of Mugilidae. In these 6 species, the relatively short 5S rDNA repeat units, generated by PCR and ranging in size from 219 to 257 bp, show a high level of intragenomic homogeneity of both coding and spacer regions (NTS-I). Phylogenetic reconstructions based on this data set highlight the greater phylogenetic and genetic diversity of Mugil cephalus and Oedalechilus labeo compared with the genera Liza and Chelon. Comparative sequence analysis revealed significant conservation of the short 5S rDNA repeat units across Chelon and Liza. Moreover, a second size class of 5S rDNA repeat units, ranging from roughly 800 to 1100 bp, was produced in the Liza and Chelon samples. Only short 5S rDNA repeat units were found in M. cephalus and O. labeo. The sequences of the long 5S rDNA repeat units, obtained in Chelon labrosus and Liza ramada, differ owing to the presence of 2 large insertion/deletions (indels) in the spacers (NTS-II) and show considerable sequence identity with NTS-I spacers. Interspecific sequence variation of NTS-II spacers, excluding the indels, is low. Southern-blot hybridization patterns suggest an intermixed arrangement of short and long repeat units within a single chromosome locus.     

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T_m > 70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners.     

Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.

Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by the monoclonal antibodies HMFG-1, HMFG-2, and SM-3. Previously isolated partial cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 20-amino acid repeat units. We now report the full sequence for PEM, as deduced from cDNA sequences. The encoded protein consists of three distinct regions: the amino terminus consisting of a putative signal peptide and degenerate repeats; the major portion of the protein which is the tandem repeat region; the carboxyl terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail. Potential O-glycosylation sites (serines or threonines) make up more than one-fourth of the amino acids. Length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus. Tandem repeats appear to be a general characteristic of mucin core proteins.