Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.

In Trypanosoma brucei, uptake of host transferrin is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor derived from the 2 expression site-associated genes 6 and 7 (ESAG6 and ESAG7). By using specific antibodies, it is shown here that T. evansi, a trypanosome species transmitted mechanically by biting flies, also expresses a transferrin receptor composed of ESAG6 and ESAG7. The cellular uptake of transferrin in T. evansi is completely inhibited with anti-T. brucei (ESAG6/7 heterodimer) antibodies. The demonstration of a functional ESAG6/7 transferrin receptor in T. evansi supports further its close relationship to T. brucei.     

The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.     

Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation.

A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites.     

Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.     

Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene

The anion/cation symporter (ACS) family is a large subfamily of the major facilitator superfamily (MFS) of transporters. ACS family permeases are widely distributed in nature and transport either organic or inorganic anions in response to chemiosmotic cation gradients. The only protein in the ACS family to which a human disease has been linked, is sialin, the proton-driven lysosomal carrier for sialic acid. Genetic defects in sialin cause a lysosomal storage disease in humans. Here we have identified a group of conserved Drosophila ACS family genes related to sialin and studied their expression patterns throughout embryogenesis. Drosophila sialin-related genes are expressed in a wide variety of tissues. Expression is frequently observed throughout various parts of the intestinal tract, including Malpighian tubules and salivary glands. Additionally, some genes are expressed in vitellophages (yolk nuclei), nervous system, respiratory tract and a number of other embryonic tissues. These data will aid the establishment of a fruitfly model of human lysosomal storage disorders, the most common cause of neurodegeneration in childhood.     

Re-expression of an inactivated variable surface glycoprotein gene in Trypanosoma equiperdum

Variable surface glycoprotein (VSG) genes in African trypanosomes are often activated by the duplicative transposition of a silent basic copy (BC) gene into an unlinked telomerically located expression site, producing an active expression-linked copy (ELC) of that gene. However, some BC genes that are already linked to a telomere are activated without apparent duplication or transposition. We have recently shown that an active VSG ELC can be inactivated in situ, apparently without rearrangement. To explain these observations it has been suggested that VSG genes that are associated with chromosome telomeres are activated by chromosome end exchanges that occur at a considerable distance upstream from the genes themselves and place them cis to a unique VSG expression element. In an attempt to test this model we derived five VSG-1 expressing variants from BoTat-2, a VSG-2 expressing variant of Trypanosoma equiperdum which carries an inactive residual VSG-1 ELC (R-ELC) as well as the active VSG-2 ELC near unlinked chromosome telomeres. We examined the fates of the VSG-2 ELC and the VSG-1 R-ELC in these variants. All five had maintained the VSG-1 R-ELC; three in a reactivated form and two in an inactive state. The latter two variants carried new, active VSG-1 ELCs: one in the site that had previously contained the VSG-2 ELC and one in a previously unidentified site. The VSG-2 ELC was lost in all five of the variants. The results are not consistent with the simple chromosome end exchange model, which predicts that the VSG-2 ELC would be inactivated but not deleted when the VSG-1 R-ELC was reactivated.     

Expansion of the complement receptor gene family. Identification in the mouse of two new genes related to the CR1 and CR2 gene family

Human cDNA probes encoding the C3b/C4b complement receptor, CR1, have been used to identify, in the mouse, two new genes which are related to CR1 but which appear to encode a different protein product. These new mouse genes, arbitrarily designated mouse genes X and Y, hybridize specifically to three different cDNA probes derived from human CR1. The degree of hybridization homology between the mouse X and Y genes suggests they are very closely related to one another; however, the chromosomal localization of the mouse X gene to chromosome 8 and the mouse Y gene to chromosome 1 indicates they are distinct gene sequences. The mRNA species detected with the X and/or Y (X/Y) sequences are approximately 2000 bases in length, but vary in both quantity and size depending upon the tissue analyzed. DNA sequence analysis of a cDNA specific for the X and Y sequences indicates the mature protein(s) will contain the 60 amino acid consensus repeat characteristic of a group of other proteins including CR1, the C3d receptor (CR2), H, C4 binding protein (C4bp), the interleukin 2 (Il 2) receptor and others. The identity of the mouse X and Y genes, and the function of the proteins which they encode, is not known; however, the small size of the mRNA and the tissue specific expression suggests they do not encode mouse CR1 or CR2 but instead encode a related protein (or proteins) which is expressed in a wide variety of mouse tissues.     

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors.

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.     

Nucleotide variability and gene expression reveal new putative genes related to seed shattering in weedy rice

Seed shattering is one of the main traits related with the domestication of cultivated rice and with the invasiveness and persistence of weedy rice. Two independent studies in 2006 have indicated that qSH1 in Japonica and Sh4 in Indica rice are major genes governing this trait. However, a wide variation of seed shattering occurs in weedy rice ecotypes from the same geographic region and even within the same ecotype. The aim of this study was to evaluate the nucleotide variability of known and putative genes related to seed shattering in cultivated rice and to identify and validate new genes related to this trait in weedy rice. The qSH1 gene was not associated with seed shattering in the evaluated genotypes. The nucleotide variability of the genes Os01g0849100 and Os08g0512400, previously identified based on a genome‐wide resequencing study, was related to seed shattering in rice. The nucleotide variability of three single nucleotide polymorphisms (SNPs) of the OsXTH8 gene, which is related to cell wall biosynthesis, was not associated with seed shattering. However, the high expression of this gene was related to the occurrence of this trait. This study evaluated jointly a series of genes involved in rice seed shattering and indicated that the genes OsXTH8, Os08g0512400 and Os01g0849100 are important for the regulation of this trait in weedy rice in addition to previously described genes. Seed shattering in weedy rice has a more complex regulation than in cultivated rice where few major genes were identified.     

The distance between Escherichia coli genes is related to gene expression levels.

It is shown that the distance between Escherichia coli genes oriented in the same direction on the chromosome is positively correlated to the expression level of the downstream gene. It is suggested that this could be a strategy to avoid interference between ribosomes translating two different genes.