An African trypanosome variant surface glycoprotein gene whose expression is not activated by duplication

A variant surface glycoprotein (VSG) of Trypanosoma brucei is encoded by a gene whose expression is not governed by duplication-transposition. There are two copies of this gene. The 5' flanking regions of the two genes are indistinguishable by restriction mapping, although each possesses approximately 5-10 Kbp of DNA which is devoid of restriction sites. All restriction enzymes tested appeared to cut genomic DNA at a uniform distance 3' of the gene. This, coupled with the observed sensitivity of both genes to BAL 31, indicates that they lie near chromosomal termini. Length variation occurs 3' of these genes in bloodstream clones and their procyclic derivatives, although the number of length variants is conserved. This suggests that length variation alone does not control VSG switching or gene expression and that constraints exist on the extent to which 3' flanking regions can vary in length.     

The role of duplication in the expression of a variable surface glycoprotein gene of Trypanosoma brucei

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to expression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well-characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accommodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.     

Characterization of the expression-linked gene copies of variant surface glycoprotein 118 in two independently isolated clones of Trypanosoma brucei.

We have analysed the gene for variant surface glycoprotein 118 in eight independent clones of Trypanosoma brucei, two of which express the 118 gene. Expression of this gene is strictly coupled to the presence of an extra copy of the gene. In both clones the expression-linked copy is transposed to the same (or a very similar) expression site elsewhere in the genome, but the length of the sequences flanking the transposed segment in the expression site differs markedly. By means of S1 nuclease protection experiments we demonstrate that the 3'-ends of the messenger RNAs for variant surface glycoproteins 118a and 118b are different, in agreement with the hypothesis that the generation of an expression-linked copy involves a recombination between the 3' segment of the basic gene copy and a homologous region present in the expression site.     

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.     

Genetics of resistance to the African trypanosomes. VII. Trypanosome virulence is not linked to variable surface glycoprotein expression

The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.     

Purification and some properties of deoxyribonuclease whose synthesis is controlled by gene 49 of bacteriophage T4.

An enzyme which specifically cleaves very-fast-sedimenting DNA of bacteriophage T4 is synthesized after infection of T4, and its synthesis is controlled by gene 49 [1,2]. This enzyme has been proved to be a DNase [2]. We have purified this DNase 3000-fold from extracts of E. coli infected with T4. The purified preparation was practically free from other DNases, and the DNase activity was not detectable in cells infected with a mutant defective in gene 49. The enzyme activity from cells infected with a temperature-sensitive mutant of gene 49 was also temperature-sensitive, suggesting strongly that gene 49 is a structural gene of the DNase. The molecular weight of the wild-type enzyme was estimated to be 50 x 10(3) by gel filtration chromatography. The purified DNase did not cleave native and denatured DNAs of T3 and T4, but cleaved renatured T3 DNA with enzymatically fragmented T3 DNA, indicating that gaps in the DNA duplex are structures susceptible to the DNase. Cleavage of the hybridized T3 DNA occurred when the fragmented DNA was phosphorylated at either the 3' or 5'-strand termini.     

Trypanosoma brucei: frequent loss of a telomeric variant surface glycoprotein gene

We have observed the loss of an inactive telomeric variant surface glycoprotein (VSG) gene that is located on a minichromosome in Trypanosoma brucei. If this is due to gene conversion, it is the third "silent" gene conversion (i.e., one that does not produce an antigenic switch) detected in 19 antigenic switches of the IsTaR 1 serodeme. This is surprisingly frequent since the immune response cannot select against the inactive gene. We estimate that 10(-1) to 10(-3) telomeric VSG gene conversions occur per generation, which is at least 100 times more frequent than antigenic switching. Since all three "silent" gene conversions involved an IsTat 5 VSG gene, the frequency may vary among telomeric VSG genes. However, the high gene conversion frequency for the 5 VSG gene does not ensure a higher antigenic switch frequency than other telomeric VSG genes for which we have probes. These results suggest that gene conversion rapidly alters the repertoire of telomeric VSG genes, possibly including those on minichromosomes, producing a continual variation in the VSG genes that are more likely to be expressed.     

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.     

Characterization of a complex glycoprotein whose variable metabolic clearance in humans is dependent on terminal N-acetylglucosamine content.

Glycoproteins can be cleared from circulation if they carry oligosaccharide structures that are recognized by specific receptors. High-mannose type and asialo complex oligosaccharides are cleared by the mannose and asialoglycoprotein receptors, respectively. This paper presents the protein and terminal saccharide characterization for nine batches of a glycoprotein developed for pharmaceutical use. Each of these batches was evaluated in human pharmacokinetic (PK) studies, and had similar terminal elimination half-lives, but the initial clearance of this glycoprotein varied between batches. The protein is lenercept, an immunoadhesin comprising the Fc domain of human IgG1 and two tumor necrosis factor (TNF) binding domains derived from the extracellular portion of the TNFR1(p55). Lenercept is manufactured in Chinese hamster ovary (CHO) cells and is extensively N-glycosylated but is devoid of high-mannose glycans. The pharmacokinetic variability between these lots only correlated with terminal N-acetylglucosamine and not with terminal galactose, sialic acid or any polypeptide related parameter. The data emphasize the need for appropriate analytical methods for the characterization of glycoproteins, especially those designed for long half-lives, and show that assessment of the content of all three terminal saccharides is sufficient to ensure consistency of their PK performance properties.     

Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda.

The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.     

Tissue-specific expression of the human alpha 1-antitrypsin gene is controlled by multiple cis-regulatory elements.

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.