Inversion in the published genetic map of linkage group VII

In the course of cloning the dim-2 gene of Neurospora crassa we found that the published map of LG VII has an inversion of a segment extending from for to un-10K. Direct physical mapping confirmed that the gene order in this region should be wc-1, for, frq, oli, un-10

Making the selectable marker bar tighter and more economical

Use of the bacterial basta resistance gene (bar) as a selectable marker in Neurospora was reported by Avalos et al (1989 Curr. Genet. 16:369-372)

One-liners

One-liner

Small scale DNA preps for Neurospora crassa

Molecular biology experiments often require preparation of small amounts of DNA from many samples. This abbreviated DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR. Starting with fresh mycelium, 20 to 40 samples can be processed in approximately two hours. Better yields (about 5 micrograms) may be obtained by suspending approximately 100 microliters of ground lyophilized mycelium in 500 microliters of isolation buffer and following the protocol starting from step 4. Spin refers to centrifugation of samples at 14,000 rpm in a microcentrifuge

Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation: Correction

Two mistakes in our article on gene replacement by gene targeting at the his-3 locus (Margolin, B.S. et al., 1997, FGN 44:34-36) have come to our attention

Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation

We report two new plasmids, pBM60 and pBM61, and procedures to efficiently generate single- copy transformants targeted to the his-3 locus in Neurospora crassa

A simple plating assay for aneuploidy in sexual progeny of Neurospora crassa, and a new allele of mei-1.

We developed a simple ascospore plating assay for aneuploidy, based on identifying disomic progeny that inherit two independently selectable mtr alleles. We validated the assay using a known meiotic mutant, mei-2. We used this assay to demonstrate that elevated frequencies of aneuploidy previously reported to be associated with reduced DNA methylation were not, in fact, due to the methylation deficiencies. A new allele of the mei-1 gene was responsible for some of the high aneuploidy

A method for finding the genetic map position of cloned DNA fragments

A method for finding the genetic map position of cloned DNA fragment

Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c Proteins Participate in the Suppression of Macrophage Apoptosis

Inhibition of macrophage apoptosis by Mycobacterium tuberculosis has been proposed as one of the virulence mechanisms whereby the pathogen avoids the host defense. The mechanisms by which M. tuberculosis H37Rv strain suppress apoptosis and escapes human macrophage killing was investigated.The screening of a transposon mutant bank identified several mutants, which, in contrast to the wild-type bacterium, had impaired ability to inhibit apoptosis of macrophages. Among the identified genes, Rv3659c (31G12 mutant) belongs to an operon reminiscent of type IV pili. The Rv3654c and Rv3655c putative proteins in a seven-gene operon are secreted into the macrophage cytoplasm and suppress apoptosis by blocking the extrinsic pathway. The operon is highly expressed when the bacterium is within macrophages, compared to the expression level in the extracellular environment. Rv3654c recognizes the polypyrimidine tract binding Protein-associated Splicing Factor (PSF) and cleaves it, diminishing the availability of caspase-8. While M. tuberculosis inhibits apoptosis by the extrinsic pathway, the pathogen does not appear to affect the intrinsic pathway. Inactivation of the intrinsic pathway by pharmacologic agents afftects M. tuberculosis and induces cell necrosis. Likewise, inactivation of PSF by siRNA significantly decreased the level of caspase-8 in macrophages.While M. tuberculosis inhibits the extrinsic pathway of apoptosis, it appears to activate the intrinsic pathway leading to macrophage necrosis as a potential exit strategy