Jan Hoeijmakers

THE ISOLATION OF PLASMIDS CONTAINING DNA COMPLEMENTARY TO MESSENGER RNA FOR VARIANT SURFACE GLYCOPROTEINS OF Trypanosoma brucei

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.

The major transcripts of the kinetoplast DNA of Trypanosoma brucei are very small ribosomal RNAs

The nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramphenicol to be predicted. The 9S RNA has little homology with other rRNAs, but a possible secondary structure is not unlike that of the 2.5-fold larger E. coli 16S rRNA. We conclude that the 12S RNA (about 1230 nucleotides) and the 9S RNA (about 640 nucleotides) are the smallest homologues of the E. coli 23S and 16S rRNAs yet observed.

Characterization of DNA from Trypanosoma brucei and related trypanosomes by restriction endonuclease digestion.

Abstract We have digested non-kinetoplast DNA from various Trypanosoma strains with restriction endonucleases and analysed the fragment distribution by one-dimensional agarose gel electrophoresis. Visual inspection of ethidium-stained gels shows differences in banding pattern between DNAs from Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum and even between different T. brucei strains, but not between three different antigenic variants derived from the same T. brucei cell. By blotting the DNA on to nitrocellulose strips the restriction endonuclease recognition sites around genes available in cloned form can be analysed by molecular hybridization and we demonstrate this for the gene which codes for one of the variant surface glycoproteins of T. brucei. The use of this method for strain classification is discussed. Renaturation analysis of T. brucei non-kinetoplast DNA shows that about 68% of this is present as single-copy DNA with a complexity of 2.5 × 107 base pairs, whereas the remainder consists of intermediate repetitive and highly repetitive DNA. The latter fraction contains a DNA which is cut by AluI into fragments of 180 base pairs, bands in CsCl at 1.690 g/cm3 and contains duplex circles heterogeneous in size and circles with duplex tails.

Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains.

Transcription of kinetoplast DNA in Trypanosoma brucei bloodstream and culture forms

Kinetoplast DNA is the unusual mitochondrial DNA of trypanosomes. In Trypanosoma brucei it consists of about lo* minicircles (0.3 km) and lo* maxicircles (6 pm) catenated into a single network. The maxicircles are probably the equivalent of mitochondrial DNA in other organisms. Here we report that a fraction of the total cellular RNA from bloodstream form and culture form T. brucei hybridizes with the maxicircle; we find no minicircle transcripts. Preferential hybridization with a 1 .8-kb2 maxicircle segment is shown to be due to two abundant RNA species, the 9 S and 12 S RNAs. After glyoxylation the apparent size of these putative rRNAs in gels is 1080 and 590 nucleofides, respectively; they are present in approximately equimolar amounts, lack poly(A) tails, their genes are adjacent, and they are transcribed from the same strand in the order 12 S-9 S. Six’additional RNA species, varying in size from 360 to 1110 nucleotides, are transcribed from segments covering 50% of the maxicircle. These transcripts are specifically retained on oligo(dT)-cellulose and presumably represent mitochondrial messenger RNAs. Recombinant DNA plasmids containing DNA

The segregation of kinetoplast DNA networks in Trypanosoma brucei

Abstract The kinetoplast DNA of Trypanosoma brucei consists of 104 minicircles (0.3 μm) and 102 maxicircles (6 μm) held together by catenation in a complex network. In electron micrographs of kinetoplast DNA spread in a protein monolayer we have identified four types of network with the appearance of different stages in network replication and segregation. We show that each network type has characteristic properties with respect to shape, size, number, and location of maxicircle loops and nicked or covalently closed character of minicircles and maxicircles. We propose a detailed model for network segregation that involves a gradual elongation of the network, followed by network cleavage. During this process the basic network structure remains unaltered, implying a complicated mechanism of minicircle rearrangements.

RNA from the insect trypanosome Crithidia luciliae contains transcripts of the maxi-circle and not of the mini-circle component of kinetoplast DNA.

We have hybridized total cellular RNA of Crithidia luciliae with the kinetoplast DNA of this organism. To allow the discrimination of DNA from mini-circles (2300 base pairs) and maxi-circles (33 000 base pairs), kinetoplast DNA was digested with restriction endonucleases and the fragments were separated by electrophoresis through an agarose gel and transferred to nitrocellulose filters by blotting. No mini-cricle transcripts were found under conditions where maxi-circle fragments showed extensive and specific hybridization. Since maxi-circle sequences are present at less than 1% of the concentration of mini-circle sequences, we conclude that mini-circles may not be transcribed at all. Predominant hybridization with the maxi-circle fragments is obtained with a segment of only 2300--2500 base pairs. The possibility that this segment codes for unusually small mitochondrial ribosomal RNAs is discussed.

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: I. Sequence evolution and transcription of the maxicircle

Abstract Kinetoplast DNA from the insect trypanosome Crithidia luciliae contains a maxicircle of 22 × 106 D. We have cleaved this DNA with endonucleases PstI, XbaI, XhoI, SstI, HaeIII, SalII, HindIII, EcoRI, and HapII and constructed a physical map of the 31 cleavage sites. The maxicircle segments hybridizing with total cellular RNA are clustered on one-half of the maxicircle; the genes for the 9 and 12 S mitochondrial (r)RNAs are located on a 1.7-kb segment. Restriction enzyme analysis indicates a sequence homology of more than 96% between the maxicircles of C. luciliae and C. fasciculata, which is not lower than that found between maxicircles of individual Trypanosoma brucei stocks. We conclude therefore that C. luciliae and C. fasciculata are one species and propose to name this species C. fasciculata and to rename C. luciliae as C. fasciculata, var. luciliae. Furthermore we show that the overall maxicircle genome organization of Crithidia resembles that of Trypanosoma.

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata. II. Sequence evolution of the minicircles.

Abstract Minicircle sequence evolution has been studied by comparison of the minicircles from Crithidia fasciculata and C. luciliae ( C. fasciculata , var. luciliae ) by restriction enzyme analysis and hybridization experiments. In contrast to the maxicircle sequence, the minicircle sequence of these trypanosomes evolves very rapidly. No conservation of restriction fragments has been observed and cross-hybridization of minicircles, minicircle fragments, and total kDNA is relatively weak. We conclude that no fragment larger than 550 bp is perfectly conserved between all minicircles of the two trypanosomes. Alterations in the minicircle fragment patterns of our stocks of trypanosomes were even apparent in a cultivation period of 1.5 to 2 years. The alterations suggest a random drift of the sequence which supports a noncodogenic function for the minicircles. Double restriction enzyme digestion experiments show that primary fragments are homogeneous with respect to cleavage by the second enzyme. This suggests that sequence rearrangements, rather than point mutations are the basis of the minicircle sequence heterogeneity.

Characterization of kinetoplast DNA networks from the insect trypanosome Crithidia luciliae

Abstract 1. We have used the restriction endonucleases EcoRI and PstI to further characterize the structural components of intact kinetoplast DNA networks from stationary phase Crithidia luciliae. 2. Endonuclease PstI cuts less than 7% of the mini-circles (the major component in the network) and appears to give a single cut in the maxi-circles, allowing the isolation of this minor component in linearized form. The molecular weight of these linearized maxi-circles, determined by electron microscopy with phage PM2 DNA as internal standard, is 22 · 106. 3. Electron micrographs of intact networks show DNA considerably longer than the mini-circle contour length (0.8 μm) either in the network or attached to the edge. This long DNA never exceeds the size of maxi-circles (10.2 μm) and it is completely removed by treatment with either endonuclease PstI or endonuclease EcoRI (which cuts the maxi-circles and about 20% of the mini-circles). We conclude that the long DNA in these networks represent maxi-circles and that long circular oligomers of mini-circles are (virtually) absent. 4. Complete removal of maxi-circles with endonucleases PstI or EcoRI has little effect on the overall structure of the network. This shows that the mini-circles are not held together on maxi-circle strings.