Alpha-helical coiled-coil structures of Trypanosoma brucei variable surface glycoproteins

We have used electron microscopy to examine purified intact variable surface glycoproteins (VSGs) from clones derived from two distinct stocks of Trypanosoma brucei. The VSG molecule from MITat 1.2 has a large elongated domain consistent with the shape of the dimeric N-terminal domain determined by X-ray analysis (see preceding paper), and a heretofore unseen short, thin fibrous tail presumed to be the C-terminal domain. Electron microscopy on DiTat 1.3, however, indicates a morphology quite distinct from that of MITat 1.2. Analysis of four VSG amino acid sequences reveals 7-fold periodicities (heptad repeats) which indicate that alpha-helical coiled-coil secondary structure elements occur in all of these VSGs, consistent with the observation of helical bundles in one VSG. These results suggest the possibility that VSG antigenic diversity may be related to a diversity in length and disposition of alpha-helical bundles and coiled-coil domains.     

Stability of expression-linked surface antigen gene in Trypanosoma equiperdum

African trypanosomes evade clearance in immune-competent hosts by periodically replacing their major surface glycoprotein with an antigenically different glycoprotein. Expression of many of these variant surface glycoproteins (VSGs) is associated with the duplication and transposition of silent basic copy genes (BCs) into unlinked genomic expression sites. The new expression-linked VSG gene copies (ELCs) are oriented with their 3' ends proximal to chromosome telomeres. Other VSG genes are activated without the production of an ELC. The 3' ends of these VSG genes are near chromosome telomeres both when they are active and when they are inactive. Recently, we have shown that activation of the VSG-1 gene in the BoTaR (Bordeaux trypanozoon antigen repertoire) serodeme of Trypanosoma equiperdum involves the duplication and transposition of a telomeric BC gene into one of at least three unlinked telomeric sites. Here we show that the VSG-1 ELC is inactivated but not eliminated in some antigenic variants derived from a VSG-1 expressor. In addition, a subsequent variant that again expresses VSG-1 has not reactivated the residual VSG-1 ELC (R-ELC), but instead contains a new, active VSG-1 ELC in an unlinked telomeric site. These results show that the simple presence of an ELC in a potential expression site is not sufficient for its expression.     

6 A-resolution X-ray structure of a variable surface glycoprotein from Trypanosoma brucei

The variable surface glycoprotein (VSG) is the predominant component of the surface coat of the African trypanosome. The expression of antigenically distinct VSGs on minor populations during infection allows the parasite to escape the host immune response. Purification of the protein is facilitated by the enzymatic release of a soluble form of VSG (sVSG) which occurs on cell lysis. The soluble form is a dimer with an approximate molecular weight of 120,000-130,000. Partial proteolysis of sVSG reveals a protease-sensitive link between an amino-terminal domain which comprises about two-thirds of the molecule, and a C-terminal domain which contains the membrane attachment site. We have obtained crystals suitable for high-resolution structural analysis from preparations of three sVSG: MITat 1.2, ILTat 1.25 and ILTat 1.22. The crystal structure of the dimer of the MITat 1.2 amino-terminal domain has been solved to 6 A resolution. We report here that the dimer is an unusual 90 A rod-like molecule composed of a helical bundle of at least four 80 A-long alpha-helices.     

Two variant surface glycoproteins of Trypanosoma brucei of different sequence classes have similar 6 A resolution X-ray structures

Antigenic variation in the African trypanosome is mediated through changes in the composition of the surface coat. By controlling expression of the major surface protein, the variant surface glycoprotein (VSG), from a repertoire of perhaps 1,000 different genes the organisms exhibit a series of antigenically distinct coats and evade the host's immune system. We have determined the 6 A resolution structure of a T. brucei variant surface glycoprotein, ILTat 1.24, using X-ray crystallography. The crystallized protein consists of the N-terminal two-thirds of the intact VSG which has a relative molecular mass (Mr) of 60,000 (60K). The structure, which includes a 90 A long alpha-helical bundle, is strikingly similar to that of the N-terminal fragment of VSG MITat 1.2 (ref. 4). Although most known VSG sequences show little similarity of primary sequence in the N-terminal domain, the similarity between the structure of a Class I (ILTat 1.24) and a Class II (MITat 1.2) VSG antigen suggests that VSGs may share a common tertiary structure.     

Instability of the Trypanosoma brucei rhodesiense metacyclic variable antigen repertoire

Trypanosoma brucei rhodesiense undergoes antigenic variation in its mammalian host by changing the glycoprotein composing its surface coat. Trypanosome clones which have the same repertoire of variable antigen types (VATs) are said to belong to the same serodeme. Tsetse flies infected with a particular serodeme extrude infective metacyclic trypanosomes which express only a restricted part of this repertoire. As the only known acquired immunity in African trypanosomiasis is VAT-specific this limitation of metacyclic VAT (M-VAT) repertoire could be important in devising a vaccine. This possibility of immunoprophylaxis could depend, however, on whether or not the M-VAT repertoire is conserved over long periods of repeated cyclical transmission and between epidemics. Studies reported here on isolates made from an East African focus of sleeping sickness over a 20-yr period suggest substantial changes in the M-VATs expressed during this time. Furthermore, we have detected change in expression of 3 M-VATs during sequential tsetse transmission of a clone in the laboratory indicating a possible instability in the organization of M-VAT genes.     

Fibronectins--adhesive glycoproteins of cell surface and blood.

A recently characterised class of adhesive, high molecular weight glycoproteins is present on the surfaces of cells, in connective tissue matrices, and in extracellular fluids. These proteins may have important roles in cellular adhesion, malignant transformation, reticuloendothelial system function, and embryonic differentiation.     

Stable dislocation configuration in the surface layer of metals

The stable dislocation configuration in the surface layer of metals was calculated according to dislo-cation energy theory. It was shown that the stable dislocation configuration was only influenced by Burger's vector, while dislocation line could fluctuate closely around the direction vertical to free sur-face in a limited range.     

Structure and expression of a cloned cDNA for human interleukin-2

A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.     

Structure of a cannabinoid receptor and functional expression of the cloned cDNA

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.     

一维与二维连续随机变量的函数的分布

现行“概率论与数理统计”教材中关于一维与二维随机变量函数的概率分布讲法上不够简练,在系统性上有欠完备,试图给出一定改进及讲法更新方面的一些建议.其中心内容是对一维随机变量单调函数的概率密度函数避免用积分表示,直接用复合函数求导方法推出;对非单调函数用划分单调区间办法给出函数概率密度的一般表达式;对二维变量试图用二维换元法给出推算二维函数分布的统一方法.     

Stable expression of the bacterial neor gene in Leishmania enriettii

Molecular genetic studies in parasitic protozoa have been hindered by the lack of methods for the introduction and expression of modified or foreign genes in these organisms. Two recent reports described the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of parasite-specific sequences. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neor) in Leishmania enriettii. A chimaeric gene containing the neor gene inserted between two alpha-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed the neor gene. One goal of this work was to analyse the sequences necessary for trans-splicing of messenger RNA, as trypanosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei and subsequently in several other trypanosomatids, including L. enriettii. Many trypanosomatid genes are arranged in tandem arrays and the intergenic sequences contain both the splice acceptor site for the addition of the spliced leader sequence and a putative polyadenylation site. Messenger RNA isolated from several different neor L. enrietti lines contained the spliced leader sequence joined to the neor gene at the position of the splice acceptor site in the alpha-tubulin intergenic sequence. The neor mRNA was also polyadenylated. Plasmid DNA is present within the drug-resistant organisms and appears to be extrachromosomal. The development of these methods allows the functional analysis of sequences necessary for trans-splicing.     

Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria

The late blood stages of the human malaria parasite, Plasmodium falciparum, carry a major surface antigen, p190, of molecular weight (Mr) 190,000. This antigenically variable protein is actively processed, first as the parasite matures and again when it is released into the blood stream and invades a new erythrocyte to initiate a cycle of growth. It elicits a strong immune response in man; all tested adult sera from endemic areas have antibodies against this protein. Our evidence indicates that purified p190 can alter the course of parasitaemia in monkeys with falciparum malaria. We have also succeeded in cloning part of the gene for p190 and expressing it in Escherichia coli. To this end we have developed a new technique, antibody select, which greatly simplifies final identification of expressing clones.     

Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor

The primary structures of two types of bovine brain substance P precursors have been determined. One precursor contains a sequence homologous to that of the amphibian peptide kassinin. This new tachykinin sequence is bounded by paired basic amino acids Lys-Arg, which suggests that, like substance P, it can be liberated from the precursor and may serve as an endogenous hormone or neuromediator in mammalian organisms.     

Expression of cloned beta-endorphin gene sequences by Escherichia coli

DNA coding for the opiate peptide beta-endorphin has been cloned into bacterial plasmids in such a way as to direct the synthesis of a hybrid beta-galactosidase/beta-endorphin protein. This hybrid protein can readily be cleaved in vitro to release biologically active beta-endorphin.