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The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.

A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a ∼140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.

In a previous work we showed a remarkable conservation of the general structure of the genome (chromo-some number and synteny) among different pathogenic species of Old World Leishmania, indicating the absence of major interchromosomal rearrangements during evolution. In the present study, we have compared the fine structure of chromo-some 5 among two of these divergent species (Leishmania major and Leishmania infantum) by means of physical mapping. Remarkably, the 42 markers jointly mapped on these two chromosomes were found in an identical order along the chromosome. This perfect colinearity of the markers is in striking contrast to the large genetic distance that separates these species and suggests that conservation of the fine-scale organisation of chromosomes may be critical in Leishmania. If this colinearity is confirmed on the whole of the chromosome set, the current systematic sequencing programme of the genome of L.major should greatly help in the development of comparative genetics between different species of Leishmania.

The genus Leishmania can be taxonomically separated into three main groups: the Old World subgenus L. (Leishmania), the New World subgenus L. (Leishmania) and the New World subgenus L. (Viannia). The haploid genome of Old World Leishmania species has been shown to contain 36 chromosomes defined as physical linkage groups; the latter were found entirely conserved across species. In the present study, we tried to verify whether this conservation of the genome structure extends to the New World species of Leishmania. 300 loci were explored by hybridization on optimized pulsed field gel electrophoresis separations of the chromosomes of polymorphic strains of the six main pathogenic Leishmania species of the New World. When comparing these New World karyotypes with their Old World counterparts, 32 out of 36 linkage groups were found conserved among all species. Four chromosomal rearrangements were found. All species belonging to the L. (Viannia) subgenus were characterized by the presence (i) of a short sequence exchange between chromosomes 26 and 35, and (ii) more importantly, of a fused version of chromosomes 20 and 34 which are separated in all Old World species. 69 additional markers were isolated from a plasmid library specifically constructed from the rearranged chromosomes 20+34 in an attempt to detect mechanisms other than a fusion or breakage: only two markers out of 40 did not belong to the linkage groups 20 and 34. On the other hand, all strains belonging to the New World subgenus L. (Leishmania) were characterized by two different chromosomal rearrangements of the same type (fusion/breakage) as above as compared with Old World species: chromosomes 8+29 and 20+36. Consequently, these two groups of species have 35 and 34 heterologous chromosomes, respectively. Overall, these results show that large-scale chromosomal rearrangements occurred during the evolution of the genus Leishmania, and that the three main groups of pathogenic species are characterized by different chromosome numbers. Nevertheless, translocations seem particularly rare, and the conservation of the major linkage groups should be an essential feature for the compared genetics between species of this parasite.

We have characterised 49 DNA probes specific for each of the six smallest chromosomes in Leishmania infantum and have examined the allocation of these probes in the molecular karyotypes of the other Old World Leishmania species Leishmania donovani, Leishmania major, Leishmania tropica and Leishmania aethiopica. These 49 probes define 6 physical linkage groups in the molecular karyotypes of various strains of L. infantum. 40 of these probes hybridise in the other Old World Leishmania species and show a remarkably conserved linkage pattern. No interchromosomal exchange nor fusion could be detected. Thus, in spite of the chromosomal size polymorphisms, the general structure of the genome seems to be conserved in the six smallest chromosomes among Old World Leishmania species. This structural genomic homogeneity should be helpful for mapping studies of any Old World Leishmania genomes.

In the present study, we have analysed the frequency and distribution of several microsatellite DNAs [(CA)n, (GGT)n and (GCA)n] in the genome of Leishmania. Hybridisation analysis on the molecular karyotypes of different Leishmania strains showed the presence of these three microsatellites on all chromosomes of the parasite. The number of microsatellite clusters appeared grossly similar among strains from different Old World complexes. However, these three microsatellite families showed an uneven distribution among heterologous chromosomes of the same strain. Moreover, restriction analysis of chromosome I in various strains of Leishmania infantum showed a strong clustering of these microsatellites in the same chromosomal region. A partial genomic library was screened with a (CA)n probe, and 21 positive clones were isolated. The sequencing of these clones confirmed the association of various microsatellites such as (CA)n, (CT)n, and (GCA)n. Finally, specific polymerase chain reaction amplification of two cloned (CA)n loci demonstrated allelic size polymorphisms among strains within L. infantum and Leishmania donovani. Most of the 34 strains analysed were found to be monoallelic, while two alleles were found in a small number of strains. The interest of these sequences for studies on ploidy and population genetics of the parasite is discussed.

The exploration of the genome of the tryponosomotid protozoan Leishmania has been difficult until recently owing to a number of obstacles, not least our ignorance of the ploidy and of the number of chromosomes (as inmany other protozoa, the latter do not condense during mitosis), the uncertainty of the species concept in these allegedly asexual protozoa and the absence of classical genetic studies. Here, Patrick Bastien, Christine Bloineou and Michel Pages discuss the advances in this field brought about by the advent of molecular biology and its techniques, with on emphasis on ploidy and genetic exchange. In particular, they discuss the data from pulsed field gel electrophoresis (PFGE). When coupled with DNA restriction analysis, PFGE constitutes a powerful tool for the direct examination o f chromosomes of protozoa.

Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as < or = 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models.

In order to clarify the interpretation of molecular karyotype polymorphisms in Leishmania, the three smallest chromosomes from five cloned strains of Leishmania infantum were identified by chromosome-specific anonymous DNA probes. The presence in the same clone of homologous chromosomes of a different size was demonstrated. The chromosome size polymorphism appeared even more dramatic, with size variations affecting up to 20% of the chromosome, and the two smallest bands in one strain being equivalent to six bands in another strain. Long-range restriction maps of five different-sized homologues of chromosome I showed the size-variation to be located to a terminal fragment in 4 out of 5 cases, and to a central fragment in one. The size-variable sequence was present on at least three other chromosomes as determined by hybridisation analysis. This suggests an instability of the subtelomeric regions such as that in Plasmodium falciparum. Lastly, the finding of several pairs of distinct-sized homologous chromosomes, together with other studies, strongly suggest that Leishmania is diploid in at least part of its chromosomal complement.

The link between nucleocytoplasmic transport and apoptosis remains controversial. Nucleocytoplasmic exchange of molecules seems indeed essential for the initiation and execution of the apoptotic programme; but inhibition of nuclear transport factors may also represent a powerful apoptotic trigger. The GTPase Ran (together with its partners), first discovered to be essential in nucleocytoplasmic transport, has multiple key functions in cell biology, and particularly in spindle assembly, kinetochore function and nuclear envelope assembly. Among the Ran partners studied, NTF2 appears to be solely involved in nucleocytoplasmic transport. Here, we localised Ran and several of its partners, RanBP1, CAS and NTF2, at the nuclear membrane in the trypanosomatid Leishmania major. Remarkably, these proteins fused to GFP decorated a perinuclear 'collar' of about 15 dots, colocalising at nuclear pore complexes with the homologue of nucleoporin Sec13. In the other trypanosomatid Trypanosoma brucei, RNAi knockdown of the expression of the corresponding genes resulted in an apoptosis-like phenomenon. These phenotypes show that Ran and its partners have a key function in trypanosomatids like they have in mammals. Our data, notably those about TbNTF2 RNAi, support the idea that active nucleocytoplasmic transport is not essential for the initiation and execution of apoptosis, and, rather, the impairment of this transport constitutes an intrinsic signal for triggering PCD.

Background Trypanosomatids exhibit a unique gene organization into large directional gene clusters (DGCs) in opposite directions. The transcription "strand switch region" (SSR) separating the two large DGCs that constitute chromosome 1 of Leishmania major has been the subject of several studies and speculations. Thus, it has been suspected of being the single replication origin of the chromosome, the transcription initiation site for both DGCs or even a centromere. Here, we have used an inter-species compared genomics approach on this locus in order to try to identify conserved features or motifs indicative of a putative function. Results We isolated, and compared the structure and nucleotide sequence of, this SSR in 15 widely divergent species of Leishmania and Sauroleishmania. As regards its intrachromosomal position, size and AT content, the general structure of this SSR appears extremely stable among species, which is another demonstration of the remarkable structural stability of these genomes at the evolutionary level. Sequence alignments showed several interesting features. Overall, only 30% of nucleotide positions were conserved in the SSR among the 15 species, versus 74% and 62% in the 5' parts of the adjacent XPP and PAXP genes, respectively. However, nucleotide divergences were not distributed homogeneously along this sequence. Thus, a central fragment of approximately 440 bp exhibited 54% of identity among the 15 species. This fragment actually represents a new Leishmania-specific CDS of unknown function which had been overlooked since the annotation of this chromosome. The encoded protein comprises two trans-membrane domains and is classified in the "structural protein" GO category. We cloned this novel gene and expressed it as a recombinant green fluorescent protein-fused version, which showed its localisation to the endoplasmic reticulum. The whole of these data shorten the actual SSR to an 887-bp segment as compared with the original 1.6 kb. In the rest of the SSR, the percentage of identity was much lower, around 22%. Interestingly, the 72-bp fragment where the putatively single transcription initiation site of chromosome 1 was identified is located in a low-conservation portion of the SSR and is itself highly polymorphic amongst species. Nevertheless, it is highly C-rich and presents a unique poly(C) tract in the same position in all species. Conclusion This inter-specific comparative study, the first of its kind, (a) allowed to reveal a novel genus-specific gene and (b) identified a conserved poly(C) tract in the otherwise highly polymorphic region containing the putative transcription initiation site. This allows hypothesising an intervention of poly(C)-binding proteins known elsewhere to be involved in transcriptional control.

Summary The LmjF01.0030 gene of Leishmania major Friedlin, annotated as ‘MCAK-like’, was confirmed as a kinesin with an internally located motor domain and termed LmjKIN13-1. Both the native form of the protein and a green fluorescent protein (GFP)-fused recombinant version were shown to be exclusively intranuclear, and, more specifically, to localize to the spindle and spindle poles. Cell cycle-dependent regulation of the protein levels was demonstrated using synchronized Leishmania cells: LmjKIN13-1 was highly abundant in the G2+M phase and present at very low levels after mitosis. Altogether, these features suggest that this protein participates in mitosis. The construction of systematic deletion mutants allowed the localization of the primary sequence regions responsible for nuclear targeting on the one hand, and for cell cycle-dependent variations on the other hand. A 42-amino-acid region of the carboxy(C)-terminal domain mediates nuclear import and could be defined as an atypical nuclear localization signal. Protein level regulation during the cell cycle was shown to also depend upon the C-terminal domain, where apparently redundant degradation signals are present. Putative degradation signals appear to be present on both sides and inside the nuclear localization signal. Further experiments strongly suggest a role for the ubiquitin/proteasome pathway in this cell cycle-dependent regulation. These data underline the importance of post-translational regulation of protein abundance in this ancestral eukaryote where transcriptional regulation seems to be rare or near absent.

Video 1Localization of Encephalitozoon cuniculi EcKLP6–GFP at the tip of the flagellum in different Leishmania cells. Cells were visualized with the 100× objective of a Zeiss Axioplan fluorescence microscope, and images were captured with a Zeiss CDD camera, under both bright and UV light.xDownload (2.69 MB ) Video 1Localization of Encephalitozoon cuniculi EcKLP6–GFP at the tip of the flagellum in different Leishmania cells. Cells were visualized with the 100× objective of a Zeiss Axioplan fluorescence microscope, and images were captured with a Zeiss CDD camera, under both bright and UV light.

Chromosome fragmentation (CF) constitutes one means of manipulating eukaryotic genomes and provides a powerful tool for examining both the structure and function of chromosomes. During the past 15 yr, CF, which is based on the use of transfection, has been widely used in yeast and mammals to elucidate the functional elements required for normal chromosome maintenance. However, in view of the relatively late development of parasite genome projects, this strategy has only been used recently in parasites. Here, we describe basic methods for CF (except telomere-mediated fragmentation) experiments and analysis in Leishmania. Current limitations of this methodology are precisely the lack of knowledge of the nature of centromeres and autonomously replicating sequences in this and other protozoa, the poor understanding of precise recombination mechanisms, as well as the fact that the deletion of unknown genes essential to parasite survival may interfere with recombination events and chromosomal rearrangements. Still, this powerful method has enriched our basic knowledge of chromosomal structure and maintenance.

Chromosome fragmentation (CF) constitutes one means of manipulating eukaryotic genomes and provides a powerful tool for examining both the structure and function of chromosomes. During the past 15 yr, CF, which is based on the use of transfection, has been widely used in yeast and mammals to elucidate the functional elements required for normal chromosome maintenance. However, in view of the relatively late development of parasite genome projects, this strategy has only been used recently in parasites. Here, we describe basic methods for CF (except telomere-mediated fragmentation) experiments and analysis in Leishmania. Current limitations of this methodology are precisely the lack of knowledge of the nature of centromeres and autonomously replicating sequences in this and other protozoa, the poor understanding of precise recombination mechanisms, as well as the fact that the deletion of unknown genes essential to parasite survival may interfere with recombination events and chromosomal rearrangements. Still, this powerful method has enriched our basic knowledge of chromosomal structure and maintenance.

The Leishmania genome project reference strain, Leishmania major Friedlin, is trisomic for chromosome 1. The complete sequence of this chromosome has revealed that the genes are grouped into two large clusters of the polycistronic type, each borne by one DNA strand and located on each side of a 1.6-kb sequence often termed the switch region. Several hypotheses concerning the role of this switch region have been put forward (region of initiation of transcription for both gene clusters, origin of replication or centromeric sequence). In the present study, we have deleted this region on the three copies of chromosome 1 by sequential targeted replacements. The absence of the switch region did not alter the mitotic stability of the three deleted chromosomes. This region therefore does not appear necessary for chromosomal replication or segregation. However, during the third targeting round, which aimed at knocking out the last switch region, a fourth copy of chromosome 1 that retained this region appeared in all clones analysed. This suggests that the persistence of this switch region is necessary for parasite survival. We then showed that the presence/absence of the switch region did not act upon the expression of a resistance marker gene inserted beforehand into the left gene cluster of the same chromosomal molecule. This result suggests that the presence of this 1.6-kb sequence is not necessary for the expression of all genes on chromosome 1.

Christine Clayton *, Mark Adams , Renata Almeida , Theo Baltz , Mike Barrett , Patrick Bastien , Sabina Belli , Stephen Beverley , Nicolas Biteau , Jenefer Blackwell , Christine Blaineau , Michael Boshart , Frederic Bringaud , George Cross , Angela Cruz , Wim Degrave , John Donelson , Najib El-Sayed , Gioliang Fu , Klaus Ersfeld , Wendy Gibson , Keith Gull , Alasdair Ivens , John Kelly , Daniel Lawson , John Lebowitz , Phelix Majiwa , Keith Matthews , Sara Melville , Gilles Merlin , Paul Michels , Peter Myler , Alan Norrish , Fred Opperdoes , Barbara Papadopoulou , Marilyn Parsons , Thomas Seebeck , Deborah Smith , Kenneth Stuart , Michael Turner , Elisabetta Ullu , Luc Vanhamme aa

A simple method for the chromosomal assignment of any DNA marker would be an important tool for the ongoing project to map the genome of the protozoan parasite Leishmania. The Leishmania chromosomes enter pulsed field gel electrophoresis (PFGE) gels under current electrophoretic conditions, but their direct identification in a given strain is hampered by their stacking in a few chromosomal bands, and by the very frequent size variations of the same chromosome among parasite strains. To overcome these problems, we determined the complete karyotypes of 12 Old World Leishmania cloned strains. This enabled us to select three of these strains that display great chromosome size polymorphisms, such that every chromosome can be individualized by a specific pattern after hybridization onto these three karyotypes. The complete resolution of the genomes of these three strains can be carried out with only three electrophoretic conditions. This makes a series of three blots sufficient for the assignment of any new marker on a particular Leishmania chromosome.

The chromosomes of the human protozoan parasite Leishmania exhibit striking size polymorphisms among different strains. To define the structural basis for these variations, we have constructed full-length restriction maps of five chromosomes of 370 to 490 kb in Leishmania infantum clone LEM1317. Rare-cutting sites for the enzymes Ase I, Dra I, Xba I, Ssp I, Spe I, and Sfi I were mapped by partial and complete digestion of either gel-purified chromosomes or total DNA. Sixty-eight anonymous DNA probes were localized on these maps, as well as the mini-exon and dihydrofolate reductase–thymidilate synthase gene probes. These maps were compared with those from other strains of L. infantum and Leishmania donovani. This showed that the distribution of the restriction sites was conserved in these two close species. Four regions involved in the size variations of three chromosomes were localized; subtelomeric sequences were responsible for size variability in three of four cases. The whole of this study takes a particular significance in the frame of the project of complete physical mapping and sequencing of the Leishmania genome.

All the physical linkage groups constituting the genome of Leishmania infantum have been identified for the first time by hybridization of specific DNA probes to pulsed field gradient-separated chromosomes. The numerous co-migrating chromosomes were individualised using the distinctive size polymorphisms which occur among strains of the L. infantum/L. donovani complex as a tool. A total of 244 probes, consisting of 41 known genes, 66 expressed sequence tags (ESTs) and 137 anonymous DNA sequences, were assigned to a specific linkage group. We show that this genome comprises 36 chromosomes ranging in size from 0.35 to -3 Mb. This information enabled us to compare the genome structure of L. infantum with those of the three other main Leishmania species that infect man in the Old World, L. major, L. tropica and L. aethiopica. The linkage groups were consistently conserved in all species examined. This result is in striking contrast to the large genetic distances that separate these species and suggests that conservation of the chromosome structure may be critical for this human pathogen. Finally, the high density of markers obtained during the present study (with a mean of 1 marker/130 kb) will speed up the construction of a detailed physical map that would facilitate the genetic analysis of this parasite, for which no classical genetics is available.

A minisatellite DNA sequence is described for the first time in Leishmania infantum. It is borne by four chromosomes and consists of an 81-bp repeat unit organised in several clusters. On chromosomes I and V of L. infantum, the clusters are tightly located in the size-variable subtelomeric regions. The organisation of this sequence may be related to that of the subtelomeric interspersed repeat sequences identified in the human genome. The sequencing of seven repeat units, some subcloned from the same cluster, allowed the definition of a consensus sequence of 81 bp, particularly G/C rich (73%). Two subfamilies were clearly defined: one exhibits a 91–95% homology with the consensus sequence; the second one comprises two monomers sharing a 91% homology but only 77% homology with the consensus sequence. The two types of monomers can be found in the same cluster. These data suggest interactions between monomers and a possible role of this sequence in the instability of these regions. Finally, restriction fragment length polymorphisms were revealed by this sequence among various strains of L. infantum. Besides allowing the detection of recombination events in the unstable regions of the chromosomes, this new marker may become a useful tool in the study of the parasite population dynamics in leishmaniasis foci.

We have resolved the molecular karyotypes of 22 Leishmania infantum strains isolated between 1980 and 1988 in a restricted geographic area and belonging to zymodemes MON-11, −29 and −33. Three strains were isolated from sandflies and all the others from human cutaneous lesions. A high degree of karyotypic homology is observed among these strains, contrasting with the highly polymorphic MON-1 strains isolated in the same area. We have analysed the time-dependent evolution of size variants of chromosomes I to V, each identified by chromosome-specific DNA probes. More evidence is given for the role of subtelomeric regions in chromosomal size variation in Leishmania for both chromosomes I and II. At the population level, the chromosomes I, II and V are present in respectively 8, 4 and 3 distinct sizes. Furthermore, and despite the small size of the sample, various combinations were observed among these different chromosomal forms. These results could be explained by the occurrence of a high rate of recurrent mutations or of genetic exchange. In contrast, only one chromosomal form was observed in individual karyotypes for the chromosomes I V. These results could tally with the hypothesis of a haploid organisation for these chromosomes and strains, or, in the frame of a diploid organisation, with the hypothesis of a predominantly automictic sexuality giving rise to 2 identical forms of the homologues in the same strain.

Leishmania is a flagellated protozoan parasite belonging to the order Kinetoplastida and to the family Trypanosomatidae. A total number of 25 species has been described,* grouped in at least 12 complexes (Lainson and Shaw, 1972, 1987; Rioux, 1986; Rioux et al., 1990). They are responsible for a wide spectrum of human diseases, mostly in the tropics and subtropics, ranging from sometimes relatively benign cutaneous disease (L. major, L. tropica, L. mexicana, L. panamensis) to mutilating mucocutaneous and diffuse cutaneous lesions (L. braziliensis, L. amazonensis, L. aethiopica), and potentially fatal visceral disease affecting the hematopoietic organs (L. donovani, L. infantum, L. chagasi). Leishmaniasis has been estimated to affect 20 million people, mainly in poor countries from both the Old and the New World (Barker, 1987).

The molecular karyotypes of 36 clones derived from 8 strains of Leishmania infantum were examined by pulsed-field gel electrophoresis. Although there appeared to be a high degree of genetic relatedness between the clones and the parent strain, a limited degree of polymorphism was noted in 50% of the clones, expressed mainly as the presence of an additional chromosome or as a chromosome size modification. Repeated subcloning in one strain showed that chromosomal rearrangements could occur during the cloning process. Chromosome homologies were examined by Southern analysis with chromosome-specific DNA probes. The results suggest a disomy for some chromosomes, but cannot exclude aneuploidy. The mechanisms possibly leading to such heterogeneity are discussed: they could involve frequent DNA amplification/deletion, and imply a 'mosaic' structure of the cultured strains or clones, with different individuals possessing differently sized versions of the same chromosomes.

High level of tyrosine protein kinase activity was found in a membrane fraction isolated from an acute myeloblastic leukemia, out of 24 leukemias of different origin investigated. The major substrate for tyrosine phosphorylation in vitro was a 58 kDA protein (p58). The phosphorylation proceeded actively at 0° C and was strongly stimulated by Mn2+ ions. Comparison by partial proteolysis of the p58 with similar phosphoproteins from a T-lymphoma line (KE37) and from lectin stimulated lymphocytes showed high similarity. The possible role of the tyrosine kinase activity in this leukemia is discussed.

The XC syncytia formation observed when ecotropic murine leukemia virus producer cells were cocultivated with XC cells was specifically inhibited by anti-MuLV gp69/71 antibodies with type- or group-specific activities. Thus, the major glycoprotein of budding viruses was probably responsible for the fusion with XC cells. Since this inhibition is specific for anti-gp69/71, it provides a simple, sensitive and convenient way to type ecotropic type C viruses and to detect anti-gp69/71. The micromethod we have developed has been applied to the detection of type-specific anti-GLV gp69/71 natural antibodies in the serum of normal mice.

A high level of tyrosine protein kinase (TPK) has been recently detected in the murine lymphoma LSTRA. The main substrate for tyrosine phosphorylation in this cell line is a Mr 55 000 protein associated with the insoluble matrix of the cell. These findings prompted the search for TPK activities in human lymphoid cells. Three human T lymphoma cell lines (i.e. Molt. 4, JM, and Ke 37) and control lymphocytes were examined. After in vitro phosphorylation of detergent insoluble extracts from human T lymphoma cells, 2 major phosphotyrosine containing proteins with Mr 55 000 and 35 000 can be detected in all three T lymphoma lines, where-as an additonal specie with Mr 78 000 is present only in the Ke 37 cell line. Similar size proteins are weakly phosphorylated in normal lymphocytes. Tyrosine phosphorylation in these proteins proceeds actively at 0° C, and is dramatically stimulated by Mn ++ ions. Partial proteolysis mappings of the Mr 55 000 phosphoproteins from murine and human lymphomas revealed a strong homology among these molecules. The function of this protein in transformed lymphocytes is discussed.