Mubashir Hanif

T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker

Abstract. The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells. Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV 35S terminator. Transformation resulted in hygromycin B-resistant clones, which were mitotically stable. Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis. The latter analysis proved both multiple- and single-copy integrations of the genes in the S. bovinus genome. A. tumefaciens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S. bovinus.

Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens

A technique was developed for transforming the ectomycorrhiza-forming basidiomycetes Suillus bovinus, Hebeloma cylindrosporum , and Paxillus involutus based on Agrobacterium tumefaciens -mediated T-DNA transfer. The selection marker employed was the Sh ble gene conferring resistance to phleomycin under control of the Schizophyllum commune GPD promoter and terminator. Transformants from all three investigated species were shown by PCR to contain the GPDScP-Shble-GPDScT construct, although the fate of the foreign DNA (integrated vs episomal, single-copy vs multi copy) could not be determined. The mycorrhiza formed between S. bovinus Bl r transformants and Pinus sylvestris did not reveal any differences from those formed with untransformed Suillus bovinus.

Targeted transgenic expression of the mutation causing Hutchinson-Gilford progeria syndrome leads to proliferative and degenerative epidermal disease

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disorder characterized by striking progeroid features. Clinical findings in the skin include scleroderma, alopecia and loss of subcutaneous fat. HGPS is usually caused by a dominant-negative mutation in LMNA, a gene that encodes two major proteins of the inner nuclear lamina: lamin A and lamin C. We have generated tetracycline-inducible transgenic lines that carry a minigene of human LMNA under the control of a tet-operon. Two mouse lines were created: one carrying the wild-type sequence of LMNA and the other carrying the most common HGPS mutation. Targeted expression of the HGPS mutation in keratin-5-expressing tissues led to abnormalities in the skin and teeth, including fibrosis, loss of hypodermal adipocytes, structural defects in the hair follicles and sebaceous glands, and abnormal incisors. The severity of the defects was related to the level of expression of the transgene in different mouse lines. These transgenic mice appear to be good models for studies of the molecular mechanisms of skin abnormalities in HGPS and other related disorders.

Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae of Suillus bovinus.

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus. Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes. Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome contains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRaclp is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity. In the predicted amino acid sequences of SbRaclp and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These domain structures suggest that in S. bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals. SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place. Cdc42p and actin were localized at the tips of S. bovinus vegetative hyphae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae. The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.

Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.

Differential Expression of A-Type and B-Type Lamins during Hair Cycling

Multiple genetic disorders caused by mutations that affect the proteins lamin A and C show strong skin phenotypes. These disorders include the premature aging disorders Hutchinson-Gilford progeria syndrome and mandibuloacral dysplasia, as well as restrictive dermopathy. Prior studies have shown that the lamin A/C and B proteins are expressed in skin, but little is known about their normal expression in the different skin cell-types and during the hair cycle. Our immunohistochemical staining for lamins A/C and B in wild-type mice revealed strong expression in the basal cell layer of the epidermis, the outer root sheath, and the dermal papilla during all stages of the hair cycle. Lower expression of both lamins A/C and B was seen in suprabasal cells of the epidermis, in the hypodermis, and in the bulb of catagen follicles. In addition, we have utilized a previously described mouse model of Hutchinson-Gilford progeria syndrome and show here that the expression of progerin does not result in pronounced effects on hair cycling or the expression of lamin B.

EXPRESSION OF MULTIPLE NEBULIN ISOFORMS IN HUMAN SKELETAL MUSCLE AND BRAIN

Nebulin is a large actin‐binding protein of the skeletal muscle sarcomere. Multiple isoforms of nebulin are produced from the 183‐exon–containing nebulin gene (NEB). Mutations in NEB cause nemaline myopathy, distal myopathy, and core‐rod myopathy.

Two alternatively-spliced human nebulin isoforms with either exon 143 or exon 144 and their developmental regulation

Nebulin is a very large protein required for assembly of the contractile machinery in muscle. Mutations in the nebulin gene NEB are a common cause of nemaline myopathy. Nebulin mRNA is alternatively-spliced so that each mRNA contains either exon 143 or exon 144. We have produced monoclonal antibodies specific for the regions of nebulin encoded by these two exons, enabling analysis of expression of isoforms at the protein level for the first time. All antibodies recognized a protein of the expected size (600–900 kD) and stained cross-striations of sarcomeres in muscle sections. Expression of exon 143 is developmentally-regulated since newly-formed myotubes in cell culture expressed nebulin with exon 144 only; this was confirmed at the mRNA level by qPCR. In fetal muscle, nebulin with exon 143 was expressed in some myotubes by 12-weeks of gestation and strongly-expressed in most myotubes by 17-weeks. In mature human muscle, the exon 144 antibody stained all fibres, but the exon 143 antibody staining varied from very strong in some fibres to almost-undetectable in other fibres. The results show that nebulin containing exon 144 is the default isoform early in myogenesis, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development.