The Neurospora crassa genome: Cosmid libraries sorted by chromosome

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome e quivalents) has been created using an improved cosmid vector pLorist6Xh, wh ich contains a bacteriophage lambda origin of replication for low-copy-numb er replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chro mosomes comprising the 42.9-Mb N. crassa genome was resolved using two tran slocation strains. Using gel-purified chromosomal DNAs as probes against th e new cosmid library and the commonly used medium-copy-number pMOcosX N. cr assa cosmid library in two independent screenings, the cosmids were assigne d to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs, the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequenc ing of an entire cosmid containing the qa gene cluster indicated a gene den sity of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cos mids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N, crassa gene sequence as a starting point for ge ne identification.