Genomic organization, chromosomal location and transcription of dispersed and repeated tubulin genes in Leishmania major

Abstract

The α and β tubulin genes of Leishmania major were cloned and used to study the genomic organization, chromosomal location and transcription of tubulin genes in L. major. The number of β tubulin isogenes was determined by hybridization of probes representing the 5′ and 3′ ends of the cloned β tubulin cDNA sequence to genomic Southern blots which showed that four complete isogenes exist on Aval fragments of size 4.4, 3.9, 1.85 and 1.7 kilobase pairs (kb). These genes are present at a relative ratio of 1:18:3:1 with the 3.9 kb fragment being tandemly repeated. The chromosomal location of each tubulin isogene was studied by purification of individual chromosomes fractionated by pulsed field gradient (PFG) gel electrophoresis. Using an improved PFG procedure, L. major contains at least 23 chromosome-sized bands some of which are present in non-stoichiometric amounts, suggesting that there are more than 23 individual chromosomes. The α tubulin genes are located on chromosome 9. The 3.9 kb β tubulin cluster and the 1.7 kb isogene are linked on chromosome 21 and two dispersed β tubulin isogenes exist on chromosomes 7 and 13. Thus, three non-allelic β tubulin loci exist in L. major. Analysis of tubulin transcripts revealed a single abundant α tubulin RNA (2050 nucleotides, nt) but three abundant β tubulin RNAs (2200, 2800, 3400 nt). The 2200 nt RNA is transcribed from the tandemly clustered β tubulin isogene on chromosome 21. The 2800 nt and 3400 nt RNAs appear to represent additional transcripts from one or both of the dispersed β tubulin isogenes on chromosomes 13 and 7.