The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, fl) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNAbinding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses.Bacteriophage Pf3 infects Pseudomonas aeruginosa PAO1 bearing the RP1 plasmid (1). It is one of a variety of filamentous viruses of Gram-negative bacteria, the best known of which are the closely related fd, fl, and M13 phages, all of which infect male strains of Escherichia coli. Other filamentous phages are Pfl (P. aeruginosa, strain K), Xf (Xanthomonas oryzae), and IKe and Ifl, both of which grow on E. coli strains bearing N and I plasmids, respectively. These viruses each contain a single-stranded circular DNA molecule encased in a slender protein sheath composed of thousands of identical protein subunits of molecular weight about 5,000 (the major coat protein) and a few copies each of a few coat proteins located at the ends of the virions (the minor coat proteins). Various comparative physical studies have shown that the packing of the DNA inside the different viruses is not the same, and also that intracellular complexes between the DNAs and their single-stranded DNA (ss DNA)-binding proteins are different (2-13).The best-characterized of these systems is that of fd (fl, M13), and it serves as the basis for comparisons (see ref. 12 for reviews). Of particular relevance to the present study is that the major coat protein subunits offd span the membrane of the infected. cell before they form a coat around the viral DNA. Their insertion into the membrane involves the processing of a signal sequence from a precursor protein. Dur-ing virus assembly, which occurs at the membrane, the viral DNA leaves its complex with its single-stranded DNA-binding protein as it becomes covered by major coat protein subunits and is extruded into the medium. The assembly and extrusion process does not cause cell lysis.In this paper, we present the results of an investigation of a region on the genome of Pf3 that encodes the DNA-binding protein and the coat protein of this virus. Comparisons of this region of DNA in Pf3 with its fd counterpart indicate close similarities in overall organization yet quite different amino acid sequences for the structural proteins themselves and the absence of a signal sequence for the major coat protein. MATERIALS AND METHODSBacteria and Bacteriophage. Pf3 bacteriophage and its host P. aeruginosa (PAO1) beari...
A numerical alteration in chromosome complement in human dermal fibroblast cultures, hyperdiploidy with a normal occurrence of tetraploidy (IVH) has been reported (Danes 1984) to be associated with some heritable single tumors including squamous carcinoma of the nasopharynx (Danes 1986). The incidence of IVH was compared in cultures derived from 65 patients with squamous carcinoma in different regions of the aerodigestive tract (ADT) and 32 clinically normal subjects without a family cancer history by 2 different assays, metaphase assay (MA) and flow cytometry (FCM).By MA, none of the 32 normals showed IVH. Of the 65 ADT patients studied, 35 had IVH and 30 did not. By FCM, there were significant differences in the FCM DNA index (p values<0.001) of “IVH” (all normals and 30 ADT patients) and IVH+ ADT patients. The per cent of cells with a DI of 2 and > I could be used to distinguish all IVH˜ from the IVH + subjects and were thus considered to be the parameters of choice in assaying IVH by FCM. None of the subjects studied showed increased in vitro tetraploidy (IVT) which has been associated with some heritable colon cancer syndromes.Irrespective of family cancer history, approximately half (35/65) of the ADT patients had IVH which has been shown to be associated with the in vivo expression of certain heritable tumors. The average age of squamous carcinoma diagnosis was earlier (mean 50 yrs) for the IVH* group than for the IVH˜ADT group (mean 72 yrs).
The incidence of hyperdiploidy in monolayer cultures of dermal fibroblasts from 88 patients with squamous carcinoma of the aerodigestive tract was compared with similar cultures from 39 normal subjects using three different techniques; 1) metaphase assay (MA), 2) flow cytometry of stationary cell cultures (FCM'), and 3) flow cytometry of proliferating dividing cell cultures (FCMd). In vitro hyperdiploidy was considered to be present (IVH+): 1) by MA if more than 4% of metaphases were altered, excluding tetraploidy; 2) by FCMS if more than 8% of cells in stationary cultures were hyperdiploid (i.e., DNA index > l), and 3) by FCMd if more than 6% of cells in logarithmic cultures were hypertetraploid (i.e., DNA index > 2). There was excellent concordance between the three assays, which assigned cell cultures from 55 of the 88 patients with aerodigestive squamous carcinoma (62%) to the in vitro hyperdiploidy positive (IVH+) category. Cell cultures from all 39 normal individuals were hyperdiploidy negative (IVH-).These data suggest that some substantial proportion of patients with aerodigestive squamous carcinoma may have a genetic predisposition for the disease, which can be identified by these assays. The flow cytometry methods were easier to carry out and may be substituted for the metaphase analyses.Key terms: Genetic predisposition, flow cytometry, monolayer culture, metaphase assay In vitro hyperdiploidy (NH) is defined as the presence of cells in culture having more than 46 metaphase chromosomes, as opposed to 92 metaphase chromosomes, or tetraploidy. It has been described in dermal monolayer fibroblast cultures of patients with some heritable tumors (4), including a family with nasopharyngeal cancer (5). We previously compared this assay with another, based on the distribution of DNA by flow cytometry of cells in stationary growth phase (FCM') (7, 8); we now compare the metaphase assay MA), FCM", and a third assay (FCMd), based on the distribution of DNA by flow cytometry of proliferating cells in logarithmic growth phase. The last assay (F'CMd) more closely approaches the proliferative cell culture conditions of the MA. Further, a concordance of all three assays would strengthen the hypothesis of a n underlying genetic disorder in patients with these tumors and enhance the potential value of IVH as a clinical marker of genetic predisposition for certain tumors. MATERIALS ANL) METHODSDermal fibroblast lines were established by standard culture methods (3) using split-thickness skin biopsies from 39 volunteers (23 males, 16 females, ages 16-88 yr) without a family tumor history and from 88 patients with various squamous carcinomas of the aerodigestive tract (50 males, 38 females, ages 18-87 yr). The cultures were maintained for 8-15 weeks prior to the assay (three to six subcultures by trypsinization after the primary explant culture) in plastic petri dishes (growth area 28 cm2) using Eagle's minimum essential medium (13) 585Subjects cm2 plastic petri dishes at an initial density of lo4 cells/ cm2, cultured fo...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.