Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

The incidence, numbers and types of Listeria monocytogenes isolated from farm bulk tank milks

Bulk tank milk from 160 producers was tested for Listeria monocytogenes at three monthly intervals over 1 year. Twenty-five producers were positive, most on a single occasion, only seven were positive on three or more of the four samplings. Listeria monocytogenes numbers were low, usually <1 ml^-1, the highest was 35 ml^-1. All isolates were serotype 1, the use of multilocus enzyme electrophoresis on representative isolates gave nine different electrophoretic types, two have been associated with listeriosis in humans or animals, a further two had only been isolated from one other source (silage or faeces), while the majority (5) were unique to milk.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Longitudinal axis thickenings in whole-mount spreads of synaptonemal complexes from Tradescantia

The whole-mount spreading technique was used to examine the chromosome complement in ten zygotene nuclei of Tradescantia ohiensis var. paludosa. Spread preparations have normal synaptonemal complex (SC) morphology. Neither kinetochores nor recombination nodules were regularly visible. Cloudlike structures (LFSs) 2–6 m in diameter were sometimes associated with SC and with unsynapsed lateral components (LCs). They differed in number and location in different nuclei. Thickenings of the lateral elements and LCs were observed in mid through late zygotene nuclei. These longitudinal axis thickenings (LATS) were distributed fairly uniformly amongst the chromosomes and occurred in both synapsed and unsynapsed regions. There was a stage-dependent increase in the number and size of LATS. They were nonrandomly distributed along the length of a chromosome, being more frequent in synapsed areas, especially near junctions with unsynapsed regions.