Chromosome complement of the fungal plant pathogen Fusarium graminearum based on genetic and physical mapping and cytological observations.

A genetic map of the filamentous fungus Fusarium graminearum (teleomorph: Gibberella zeae) was constructed to both validate and augment the draft whole-genome sequence assembly of strain PH-1. A mapping population was created from a cross between mutants of the sequenced strain (PH-1, NRRL 31084, originally isolated from Michigan) and a field strain from Minnesota (00-676, NRRL 34097). A total of 111 ascospore progeny were analyzed for segregation at 235 loci. Genetic markers consisted of sequence-tagged sites, primarily detected as dCAPS or CAPS (n = 131) and VNTRs (n = 31), in addition to AFLPs (n = 66) and 7 other markers. While most markers exhibited Mendelian inheritance, segregation distortion was observed for 25 predominantly clustered markers. A linkage map was generated using the Kosambi mapping function, using a LOD threshold value of 3.5. Nine linkage groups were detected, covering 1234 cM and anchoring 99.83% of the draft sequence assembly. The nine linkage groups and the 22 anchored scaffolds from the sequence assembly could be assembled into four chromosomes, leaving only five smaller scaffolds (59,630 bp total) of the nuclear DNA unanchored. A chromosome number of four was confirmed by cytological karyotyping. Further analysis of the genetic map data identified variation in recombination rate in different genomic regions that often spanned several hundred kilobases.

A direct method for the preparation of 2-hydroxyethoxymethyl derivatives of guanine, adenine, and cytosine.

Alkylation of 2-chloro-6-iodopurine with iodomethyl [(trimethylsilyl)oxy]ethyl ether at -63 degrees C and subsequent treatment of the 9-substituted chloroiodopurine with K2CO3 in aqueous dioxane at 25 degrees C and then with NH3 under pressure at 150 degrees C provided 9-[(2-hydroxyethoxy)methyl]guanine (1a), a potent antiviral agent against Herpes simplex virus type 1, in excellent yield. Its monophosphate (1g), which is enzymatically produced from 1a in the virus-infected cell, was also synthesized. 6-Chloropurine and 4-(methylthio)pyrimidin-2-one anions were similarly alkylated with iodomethyl [(trimethylsilyl)oxy]ethyl ether, and the products (1f and 2b) were transformed by treatment with methanolic NH3 at 110 degrees C into 9-[(2-hydroxyethoxy)methyl]adenine (1b) and 1-[(2-hydroxyethoxy)methyl]cytosine (2a), respectively. The syntheses of these analogues, heretofore difficult to prepare by a simple procedure, have been conveniently accomplished.

Monitoring fusion protein cleavage by capillary electrophoresis.

Capillary electrophoresis was successfully utilized to monitor the proteolytic cleavage of two fusion proteins; maltose binding protein-sugarcane mosaic potyvirus coat protein and maltose binding protein-paramyosin. The course of proteolysis by factor Xa was monitored with high sensitivity and virtually in real time. Capillary electrophoresis conditions were optimized to clearly resolve the fusion proteins, factor Xa, lysis products and buffer components. Rapid elucidation of time to complete digestion was possible and fate of released proteins could also be examined.

The effect of impact on the marrow pressure of long bones in vitro.

Intact fresh sheep tibiae were subjected to non-destructive impact loads while maintained in an environment like that encountered in life. Under a maximum load of 2.5 k N, and loading rate of 500N ms-1, marrow pressures no higher than 50 k Pa were recorded. Such low pressures would provide only a trivial amount of hydraulic strengthening, and are consistent with results from other workers carried out at lower rates of loading.

Swine immunity to selected parasites.

Swine parasitism exerts a significant economic impact worldwide. In the United States, the greatest losses are due directly or indirectly to the costs of zoonotic parasitisms. Three of the six most common foodborne parasitic diseases of humans in the United States are associated with pork consumption. These include toxoplasmosis, taeniasis or cysticercosis (caused by the pork tapeworm Taenia solium), and trichinellosis. Toxoplasmosis is of particular concern because of the fulminating disease that occurs in immunocompromised people. Generalizations and extrapolations of information derived from rodent and human studies, to swine parasitisms, are complicated by immunological differences between the hosts, and by the diverse biological characteristics of internal and external parasites studied. Swine studies thus far reported have demonstrated that protective immunity to helminth infection involves both cellular and humoral mechanisms, with antibodies and antibody-mediated responses playing important roles in preventing establishment of newly acquired larvae. Protection against protozoan parasites is primarily by cell-mediated strategies, whereas protective immunity to arthropod infestation is primarily through humoral mechanisms, principally those associated with type 1 hypersensitivity.

Elimination of femoral rotation in clinical testing for varus/valgus laxity of the knee.

Clinical testing of varus/valgus laxity of the knee joint has been shown to have a large interobserver variation. Part of the reason for this is the tendency for the femur to rotate during the performance of classical testing, leading to misinterpretation of the degree of joint opening. A simple adjustment in the examination technique is described which eliminates this problem, and the biomechanical principles responsible for its efficacy are outlined.

Rheology of bovine bone marrow.

The viscosity of bovine bone marrow was measured using samples taken from proximal and distal sites of five radii. The viscosity was found to be independent of shear rate and temperature above 37 degrees C (distal samples) and 42 degrees C (proximal samples). The viscosity of all samples fell to a lower limit of 0.04 Pas above these temperatures, irrespective of the treatments used. Below them the measured viscosity of the proximal marrow was some ten times that of the distal marrow at 35 degrees C and some 15 times that of the distal marrow at 30 degrees C. Below 30 degrees C the proximal marrow solidified. Distal marrow remained liquid to below 20 degrees C.

Comparison of influenza B/Hong Kong virus infections among infants, children, and young adults.

An influenza B/Hong Kong viral epidemic was monitored by surveillance of respiratory illness in three different age groups. Prospective viral monitoring of febrile respiratory illness was a useful mirror of the epidemiologic behavior of influenza in the community. Influenza B virus infection in infants and young children was distinguished by high fever and respiratory symptoms and was occasionally associated with otitis media. In older children and young adults, systemic and gastrointestinal complaints were more prominent. Of young children experiencing their first infection, a hemagglutination-inhibiting antibody response was seen in only one-third. Eleven children who received inactivated B/Hong Kong virus vaccine three years earlier were not protected from clinical infection but exhibited an anamnestic serum antibody response.

Canine kidney cell line for isolation of respiratory viruses.

By means of a continuous canine kidney cell line (MDCK), influenza viruses were rapidly isolated from specimens collected from patients with respiratory disease. The cell line proved more sensitive than either eggs or rhesus monkey cells for currently circulating influenza A and B strains. Influenza viruses caused a distinct cytopathology within 5 days of inoculation if trypsin-ethylenediamine-tetraacetic acid was incorporated into the medium. Sufficient hemagglutinin was produced on the initial tissue culture passage to allow direct identification of isolates by hemagglutinin inhibition tests. A variety of other respiratory viruses replicated in MDCK, and over a 10-month period 211 of 600 specimens (35%) yielded viruses.

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.