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  • volume 13 Number 11 1985 Nucleic Acids Research

    The molecular karyotype of Leishmania major and mapping of a and 0 tubulin gene families tomultiple unlinked chromosomal loci

    Terry W.Spithill and Nicholas Samaras

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

    Received 1 April 1985; Revised and Accepted 13 May 1985

    ABSTRACTThe arrangement of tubulin genes in the genome of the protozoan parasite

    Leishmania major was studied by genomic Southern blot analysis and mapping ofgenes to chromosomes fractionated by pulsed field gradient gel (PFG) electro-phoresis. a-tubulin genes exist as a tandem array of 2.4 kb PstI fragments,p-tubulin genes are found as a tandem array of 3.9 kb Aval or Pvul fragments,but additional genes are also found on other genomic DNA fragments. Chromo-some-sized DNA molecules released from promastigotes of L.major were fractiona-ted into at least 17 chromosome bands of approximate size 400-4000 kb by PFGgel electrophoresis. Some bands may be present in non-equimolar amountssuggesting that there may be more than 17 chromosomes. All a-tubulin geneswere localized to a single band (chromosome 7). p-tubulin genes were localizedto four bands (chromosomes 6, 10, 16 and 17). This shows that the a- and p-tubulin gene families are unlinked in L.major. There is a single chromosomallocus for the a-tubulin tandem array whereas p-tubulin genes exist both as atandem array and as dispersed genes at four chromosomal loci.


    The arrangement of tubulin genes in the protozoan parasite Leishmania

    1s novel among eukaryotes. With most organisms, a- and p-tubulin genes exist

    as multiple copies which are dispersed in the genome (1-4). In Trypanosoma

    brucei, these genes exist as a tandem array of alternating a- and p-tubul1n

    gene pairs (5). In contrast, Leishmania possess separate tandem repeat arrays

    of a- and p-tubulin genes (6-8). This unique arrangement in Leishmania may

    reflect a gene organization which has evolved to allow the efficient control

    of gene expression in an organism which exhibits rapid changes in the levels

    of tubulins expressed during Its life cycle (9).

    An interesting feature of the tubulin system in Leishmania is the flexi-

    bility that exists in the type of control of tubulin gene expression exhibited

    by different species during differentiation from the flagellated promastigote

    form to the non-flagellated amastigote form. In L.enriettH , the expression

    of the single size class of a and p tubulin mRNA is controlled at the level

    of mRNA accumulation (10). In L.mexicana amazonensis, the expression of the

    IRL Press Limited, Oxford, England. 4 1 5 5

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    single size class of a tubulin mRNA 1s translationally controlled: 1n

    contrast, three size classes of p tubulin mRNA exist and, although the total

    amount of p tubulin raRNA 1s constant during differentiation, the level of

    accumulation of each of these three size classes 1s differentially regulated

    (11,12). These results suggest that multiple p tubulins exist In

    ensis and show that the control of tubulin expression in species of Le1shman1a

    is both flexible and complex. Our understanding of the molecular basis of

    this flexibility in control will require a comparative analysis of the chromo-

    somal arrangement of a and p tubulin genes in different Leishmania. This 1s

    necessary in order to answer several Important questions concerning tubulin

    gene arrangement such as the number of non-allel1c tubulin genes, whether

    the a and p tubulin gene families are linked, whether dispersed genes also

    exist and whether these dispersed genes are located on the sane chromosome

    as the tandem repeat arrays.

    Recently, the technique of pulsed field gradient (PFG) gel electrophoresis

    has permitted the fractionation of chromosome-sized DNA molecules from yeast,

    trypanosones and other kinetoplastid protozoa Including L.tropica (13-16).

    The localization of tubulin genes 1n L.tropica to two distinct chromosomal

    size classes was shown by Southern blotting and probing with an ap-tubulin

    probe from T.brucei (16). However, this experiment could not map individual

    a- or p-tubulin genes to specific chromosomes, show the linkage of the two

    gene families or reveal the number of loci for a- and p-tubulin.

    Here, we report our initial studies on the chromosomal arrangement of

    tubulin genes 1n L.major. Using genomic Southern blot analysis, we show that

    a and p tubulin genes exist as separate tandem repeat arrays and that p-tubul1n

    genes also exist as dispersed gene copies. By PFG gel electrophoresis, we

    find that a cloned line of L.major contains at least 17 chromosomes and have

    mapped the chromosomal location of the multiple a- and p-tubulin genes. The

    results show that the cr-and p-tubul1n gene families are unlinked, that there

    1s a single locus for or-tubul1n but multiple dispersed lod for the p-tubul1n




    The cloned line of L.major. LRC-L137/7/V121 (termed V121), was isolated

    and grown 1n NNN medium as described (17).

    Isolation and analysis of genomic DNA

    Genomic DNA was isolated froa promastigotes of V121 as described (18).


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    DMA (1 pg) was digested for 16h at 37C with 10 units of restriction enzyme

    under the conditions recommended by the supplier. Digested DNA was fraction-

    ated by electrophoresis In a 0.7X agarose gel 1n TBE buffer (19). The gel

    was processed for Southern blotting, the DNA was transferred to a nitrocellu-

    lose filter and prepared for hybridization as described (19). Filters were

    hybridized at 42C for 20-24h 1n hybridization medium (19) containing 1-5 x

    10 cpm/ml of nick-translated (20) probes, washed In 2 x SSC, 0.1* SDS at

    65C for 4h, dried and exposed to X-ray f1la for 2-4 days.

    PFG gel electrophoresis of chromosomes

    Promastigotes of strain V121 were harvested, washed and suspended 1ng

    phosphate buffered saline (PBS) at a cell density of 3 x 10 /ml. Following

    addition of an equal volume of molten 2% low melting agarose (Seakem) 1n PBS

    at 37C, the noHen mixture was embedded in the wells of a slot holder (1.6

    x 5 x 5 m slots) and allowed to set. The agarose inserts were placed 1n

    lysing buffer (0.5 M EDTA, 0.01 M Tris, IX Sarkosyl, pH 9.5 at 42C) contain-

    ing 2 mg/ml Proteinase K (Merck) and Incubated at 42C for 48h. The Inserts

    were stored at 4C 1n lysing buffer. For fractionation, an agarose Insert

    was sliced Into four pieces and Incubated for lh In 0.9 x TBE buffer. Each

    piece was placed 1n the well of a 1.5% agarose (Seakem HE) gel 1n 0.9 x TBE,

    the well was filled with 1% low melting agarose 1n 0.9 x TBE and subjected

    to PFG gel electrophoresis (13). The samples were subjected to 200V (lOV/ca)

    for 2h In the north-south direction (top-bottom) and then fractionated for

    21-24h with 40-80 sec pulses, 250V (12.5V/cn) in the north-south direction

    and 100V (5V/cm) in the east-west (left-right) direction at 18C-23C. Follow-

    ing fractionation, the gel was stained with ethidlun bromide (1 pg/ral) for

    16h and photographed. Chromosomal DNA was blotted from the gel onto nitro-

    cellulose filters as described above. The approximate molecular weights of

    the chronosoaes of L.major were estimated relative to the reported approximate

    sizes of the yeast chromosomes which were prepared from S.cerevisiae strain

    J69bl as described (13). Due to the uncertainty 1n the sizes of the yeast

    chromosomes and the nature of the fractionation observed, the precise size

    of the L.major chromosomes 1s uncertain.


    Genoaic structure of tubulin genes

    Genonic DNA was Isolated from promastigotes of the cloned line V121 of

    L.major LRC-L137, digested with various restriction endonucleases, fraction-

    ated by agarose gel electrophoresis and blotted onto nitrocellulose filters.


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    Figure 1. Genomic Southern blot analysis of tubulin genes. Genomic DNA frompromastigotes of L.major LRC-L137/7/V121 was digested with PstI(lanes 1,4), Aval (lane 2) or Pvul (lane 3), fractionated, blottedand hybridized to probes for a-tubulin (lane 1) or p-tubulin (lanes2-4). The probes were plasmid DNA Isolated from clones pP94/l andpP121/4 which contain cloned cDNA sequences for a- and p-tubulinrespectively. These+clones were isolated from cDNA libraries syn-thesized from poly A mRNA isolated from L.major V121 as will bedescribed elsewhere using cloned a- and p-tubulin cONA sequencesfrom chicken (1). The sizes of the genoraic DNA fragments homologousto the probes is shown beside the lanes 1n kb. In lane 3, thehybridizing fragments are of size 3.9, S.8, 7.9, 9.2 and >25 kb.

    The filters were hybridized to nick-translated probes derived from plasmid

    cDNA clones pP94/l and pP121/4 which contain cDNA inserts corresponding to

    genes for a- and p-tubulin of L.aajor, respectively. These cDNA sequences

    were isolated from an L.major cDNA library using cloned probes for the or-

    and p-tubulin genes of chicken (1) (legend, Fig. 1). The 1.5 kb insert