At least two transposed sequences are associated in the expression site of a surface antigen gene in different Trypanosome clones

The expression of several trypanosome surface antigen genes proceeds by duplication of a basic copy (BC) of the gene and transposition of the expression-linked copy (ELC) into an expression site. This site, which seems to be the same for different genes of the same repertoire, is located near a chromosome end. In the AnTat 1.1 antigen gene expression site, the ELC is found associated with another sequence that we have called the “companion.” We found that this companion is the transposed copy of another sequence also located in an unstable DNA terminus, and that it is conserved in the expression site of AnTat 1.10 and AnTat 1.1B, two clones successively derived from AnTat 1.1. The companion sequence is not part of the surface antigen gene, but we may infer from extensive homologies with another ELC sequence (IoTat 1.3, J. E. Donelson, personal communication) that it represents a 5′ residual fragment of a former ELC. In three other AnTat 1.1-like clones, the companion sequence was not found associated with the ELC. It is concluded that the expression-linked duplicative transposition of variable antigen genes is a flexible mechanism, which can apply to variably sized stretches of the same BC.     

Gene conversion as a mechanism for antigenic variation in trypanosomes

Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs--AnTat 1.1, 1.10, and 1.1B--are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.     

Inactivation and reactivation of a variant-specific antigen gene in cyclically transmitted Trypanosoma brucei.

In Trypanosoma brucei, the activation of the variant-specific antigen gene AnTat 1.1 proceeds by the synthesis of an additional gene copy, the AnTat 1.1 ELC, which is transposed to a new location, the expression site, where it is transcribed. Using the AnTat 1.1 variant to infect flies, we investigated the fate of the AnTat 1.1 ELC during cyclic transmission of T. brucei. We show here that the AnTat 1.1 ELC is conserved in procyclic trypanosomes, obtained either from the midgut of infected Glossina or from cultures, and in metacyclic trypanosomes, although the AnTat 1.1 serotype is not detected among metacyclic antigen types. This same AnTat 1.1 ELC, which is thus silent as the parasite develops in the insect vector, can be reactivated without duplication during the first parasitemia wave following cyclical transmission. This re-expression of the conserved ELC accounts for the early appearance of the 'ingested' antigenic type after passage through the fly.     

Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.     

Trypanosoma brucei: the extent of conversion in antigen genes may be related to the DNA coding specificity

The boundaries of gene conversion in variant-specific antigen genes have been determined in six clones of Trypanosoma brucei. In each clone, antigenic switching involved interaction between two telomeric members of the AnTat 1.1 multigene family, which share extensive homology throughout their coding regions. All conversion events occurred by substitution of faithful copies of donor sequences. Conversion endpoints were nonrandomly distributed. In four clones, the 5' conversion limit was near the antigen translation initiation codon, while in three clones, the 3' conversion limit was located at the "hinge" between the two major antigen domains. In one case, two segmental conversions were involved in antigen switching. These observations reveal that antigen gene conversion can occur without generating point mutations, and suggest that postrecombinational selection may impose a limit on the number of possible rearrangements within antigen genes.     

Trypanosome variant surface glycoprotein genes expressed early in infection

We have studied further the genes for trypanosomal variant surface glycoproteins expressed during a chronic infection of rabbits with Trypanosoma brucei, strain 427. We show that there are three closely related chromosomal-internal isogenes for VSG 121; expression of one of these genes is accompanied by the duplicate transposition of the gene to a telomeric expression site, also used by other chromosome-internal VSG genes. The 3' end of the 121 gene is replaced during transposition with another sequence, also found in the VSG mRNAs of two other variants. We infer that an incoming VSG gene duplicate recombines with the resident gene in the expression site and may exchange ends in this process. The extra expression-linked copy of the 121 gene is lost when another gene enters the expression site. However, when the telomeric VSG gene 221 is activated without duplication the extra 121 gene copy is inactivated without detectable alterations in or around the gene. We have also analysed the VSG genes expressed very early when trypanosomes are introduced into rats or tissue culture. The five genes identified in 24 independent switching events were all found to be telomeric genes and we calculate that the telomeric 1.8 gene has a 50% chance of being activated in this trypanosome strain when the trypanosome switches the VSG that is synthesized. We argue that the preferential expression of telomeric VSG genes is due to two factors: first, some telomeric genes reside in an inactive expression site, that can be reactivated; second, telomeric genes can enter an active expression site by a duplicative telomere conversion and this process occurs more frequently than the duplicative transposition of chromosome-internal genes to an expression site.     

Translocation alters the activation rate of a trypanosome surface antigen gene.

We report here the characterization of the gene coding for AnTat 1.13, a very late variable antigen type (VAT) from Trypanosoma b. brucei. This gene is chromosome-internal and it is activated by the duplicative mechanism. Like in another case of late VAT expression (1), its expression-linked copy (ELC) is flanked by "companion" sequences. It was possible to convert the late expression of this VAT into an early one, by changing the location of the gene in the genome. This has been achieved by selecting an AnTat 1.6 clone among heterotypes arising in the AnTat 1.13 cloned population. Indeed, this particular derivation leads to the conservation of the AnTat 1.13 ELC as a new telomeric member of the gene family, and this conserved ELC (or ex-ELC) appears to be preferentially activable. The telomeric position and other factors possibly involved in early or late antigen gene expression are discussed; in this respect, we propose that some antigen genes are rarely activated because their duplicative transposition requires the presence, in the expression site, of "companion" sequences only shared by a limited number of other genes.     

Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei

We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.     

Analysis of the DNA and RNA changes associated with the expression of isotypic variant-specific antigens of trypanosomes.

Using specific (32P) labelled cDNA probes, we compared the mRNAs and the genomic DNA sequences coding for the synthesis of two pairs of serologically related variant-specific antigens (VSAs) of trypanosomes: AnTat 1.1 and AnTat 1.1b, both from the strain 1125 of T.b.brucei and AnTat 1.8 and LiTat 1.6 from T.b.brucei and T.b. gambiense, respectively. Within each pair, large similarities were observed in the coding sequence, except in the 3' region which appears to be highly variable. However, a low level of cross-hybridization can be detected between all sequences, in the 3' region only. The expression of these VSAs is linked to a similar duplication-transposition mechanism. The insertion locus of the transposition unit is the same both in AnTat 1.1 and AnTat 1.1b DNAs. In both pairs, the transposition unit seems to comprise at least about 200 bp upstream of the 5' extremity of the coding sequence. The significance of these results, regarding the structure and synthesis of the VSAs, is discussed.     

The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Antigenic variation by Borrelia hermsii occurs through recombination between extragenic repetitive elements on linear plasmids

The relapsing fever agent Borrelia hermsii undergoes multiphasic antigenic variation through gene conversion of a unique expression site on a linear plasmid by an archived variable antigen gene. To further characterize this mechanism we assessed the repertoire and organization of archived variable antigen genes by sequencing approximately 85% of plasmids bearing these genes. Most archived genes shared with the expressed gene a 相似文献   

Molecular Cloning of a Novel Human Acid Phosphatase Gene (ACPT) That Is Highly Expressed in the Testis

Acid phosphatases are enzymes capable of hydrolyzing orthophosphoric acid esters in an acid medium. Prostatic acid phosphatase has served as a tumor marker for metastatic prostate cancer for many years. We have cloned a new human acid phosphatase gene (named testicular acid phosphatase, ACPT), which is expressed mainly in testis and to a lower extent in the prostate, trachea, and other tissues. This gene maps to chromosome 19q13.4, in an area that harbors many cancer-related genes. The testicular acid phosphatase gene is composed of 11 exons, and the protein is predicted to have a luminal domain, a transmembrane domain, and a cytoplasmic domain. The N-terminal end of the protein encodes a signal peptide. The protein has approximately 50% homology with both the prostatic and the lysosomal acid phosphatases, and the position of the cysteine residues, the N-glycosylation sites, and the histidine catalytic site are conserved among the three proteins. The testicular acid phosphatase gene is up-regulated by androgens and is down-regulated by estrogens in the prostate cancer cell line LNCaP. Our preliminary results indicate that this gene exhibits a lower level of expression in testicular cancer tissues than in their normal counterparts.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Controlled Integration into the Lactococcus Chromosome of the pCI829-Encoded Abortive Infection Gene from Lactococcus lactis subsp. lactis UC811

The phage insensitivity gene of lactococcal plasmid pCI829 which encodes an abortive infection defense mechanism (Abi) was inserted into the Lactococcus lactis subsp. lactis CH919 chromosome by utilizing the integration plasmid pCI194, which contains 4.2 kb of homology with the conjugative transposon Tn919. Chloramphenicol-resistant transformants expressed phage insensitivity to the prolate-headed phage c2 and the small isometric-headed phage 712, and hybridization analysis indicated that transformants contained pCI194 integrated in single copy. The level of phage insensitivity expressed by the transformants was reduced from that observed when the abi gene was located on a replicating plasmid, as determined by plaque assay and burst size analysis. Amplification of the integrated structure after growth in increased concentrations of chloramphenicol resulted in an increase in the expression of phage insensitivity. Hybridization analysis revealed that while pCI194 was stably maintained in an integrated state over 100 generations in the absence of selective pressure, the ability to express phage insensitivity was lost. Hybridization analysis also revealed that DNA flanking the abi gene contains homology to the CH919 chromosome.     

The effect of heterologous insertions on gene conversion in mitotically dividing cells in Drosophila melanogaster

We examined the influence that heterologous sequences of different sizes have on the frequency of double-strand-break repair by gene conversion in Drosophila melanogaster. We induced a double-strand break on one X chromosome in female flies by P-element excision. These flies contained heterologous insertions of various sizes located 238 bp from the break site in cis or in trans to the break, or both. We observed a significant decrease in double-strand-break repair with large heterologous insertions located either in cis or in trans to the break. Reestablishing the homology by including the same heterologous sequence in cis and in trans to the double-strand break restored the frequency of gene conversion to wild-type levels. In one instance, an allelic nonhomologous insertion completely abolished repair by homologous recombination. The results show that the repair of a double-strand break by gene conversion requires chromosome pairing in the local region of the double-strand break.