The influence of socio-economic and surveillance characteristics on breast cancer survival: a French population-based study.

Survival data on female invasive breast cancer with 9-year follow-up from five French cancer registries were analysed by logistic regression for prognostic factors of cancer stage. The Kaplan-Meier method and log-rank test were used to estimate and compare the overall survival probability at 5 and 7 years, and at the endpoint. The Cox regression model was used for multivariate analysis. County of residence, age group, occupational status, mammographic surveillance, gynaecological prevention consultations and the diagnosis mammography, whether within a screening framework or not, were independent prognostic factors of survival. Moreover, for the same age group, and only for cancers T2 and/or N+ (whether 1, 2 or 3) and M0, the prognosis was significantly better when the diagnosis mammography was done within the framework of screening. Socio-economic and surveillance characteristics are independent prognostic factors of both breast cancer stage at diagnosis and of survival. Screening mammography is an independent prognostic factor of survival.

Streptothricin resistance as a novel selectable marker for transgenic plant cells.

Streptothricins are known as antimicrobial agents produced by Streptomyces spp. Bacterial resistance to streptothricin is mediated by specific enzymes exhibiting an acetyltransferase activity which renders the drug non-toxic for bacteria. The nucleotide sequence of several streptothricin resistance genes from bacteria have been described. Certain cells of eukaryotic parasites (such as Ustilago maydis or Leishmania spp.) are sensitive to streptothricin and the introduction of the bacterial resistance gene sat2 renders them resistant. We show that numerous species of plants are sensitive to low concentrations of streptothricin. Moreover, introduction of the bacterial resistance gene sat3 under the control of the 35S cauliflower mosaic virus promoter protects these cells from the toxic action of streptothricin. Therefore, sat3-mediated streptothricin resistance appears to be a promising selective marker for genetic manipulation of plant cells.

Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants.

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5' deletions of the ORF13 promoter fused to the beta-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected.

Nucleotide sequence of the bacterial streptothricin resistance gene sat3.

The nucleotide sequence of the sat3 gene which encodes resistance of enteric bacteria to the antibiotic streptothricin is reported. A protein with a molecular mass of about 23 kDa is expressed from this gene. The sat3 gene is not obviously related to any one of the streptothricin resistance determinants identified so far among Gram-negative or Gram-positive bacteria.

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression.

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA--which we refer to as rol alpha, beta and gamma--were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5' non-coding region of rol alpha and rol beta was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol beta in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol alpha-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.

A new open reading frame, encoding a putative regulatory protein, in Agrobacterium rhizogenes T-DNA.

We report the presence of an open reading frame, named ORF13a, encoding a putative regulatory protein on the T-DNA of Agrobacterium rhizogenes 8196 Ri plasmid. Homologous ORFs are present at the same location in two other types of Ri plasmids. We present evidence that ORF13a is transcriptionally active. Expression of ORF13a was investigated by analysis of glucuronidase (GUS) activity in transgenic tobacco containing an ORF13aGUS fusion. The gene fusion was expressed at higher level in roots than in leaves. The putative protein encoded by ORF13a has an isoelectric point of 11.55 and carries SPXX repeated motifs suggesting a possible regulatory function for this gene.

A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196.

An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA right-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on an Agrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named 'T-DNA transfer stimulator sequence' (TSS).

Agrobacterium rhizogenes pRi8196 T-DNA: mapping and DNA sequence of functions involved in mannopine synthesis and hairy root differentiation.

This paper presents the map and DNA sequence analysis of pRi8196 transferred DNA (T-DNA) genes encoding root-inducing and mannopine synthesis functions. A canonical 24-base-pair border repeat as well as two "pseudoborders" are present at the functional right T-DNA border. To the left of this border are homologs of the mas1' and mas2' genes of TR pRiA4. Next to these are five open reading frames (ORFs) homologous to ORFs 10-14 of TL of pRiA4. ORFs 10-12 (rolA, rolB, and rolC) are less related to their pRiA4 homologs than are the other large ORFs analyzed here. In contrast to T-DNA genes of pRiA4, pRi8196 T-DNA ORFs 11 and 12 (rolB and rolC) are sufficient to induce hairy roots on carrot disks.

The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene.

The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.

Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco.

The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 TL-DNA was studied by using the beta-glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolB-GUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.

Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies.

Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (TL-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the TL-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.

Cloning and preliminary characterization of the streptothricin resistance determinants of the transposons Tn1825 and Tn1826.

Streptothricin resistance determinants have been cloned from the transposons Tn1825 and Tn1826 to the vector plasmid pUC8. The recombinant plasmids were characterized with respect to their physical structure. The resistance properties of their hosts were characterized with respect to the minimal inhibitory concentration of streptothricin and the activity of the streptothricin acetyltransferase. The proteins encoded by the cloned resistance determinants were analysed in a maxicell system. The results of our investigations suggest that the resistance to streptothricin is mediated by the activity of a streptothricin acetyltransferase with both, Tn1825 and Tn1826. However, the resistance determinant of Tn1825 is more complex than Tn1826 and additional functions involved in realizing the resistance phenotype must be recognized.

Relationships among the streptothricin resistance transposons Tn1825 and Tn1826 and the trimethoprim resistance transposon Tn7.

The streptothricin resistance transposons Tn1825 and Tn1826 are closely related, based on physical and genetic characteristics, to the trimethoprim resistance transposon Tn7. These transposons may be considered to be members of a transposon family sharing in common the transposition functions and a basic streptomycin/spectinomycin resistance determinant but differing from one another with respect to particular additional resistance genes inserted to the left of the aadA gene.

Physical map of the T-DNA region of Agrobacterium rhizogenes strain NCPPB2659.

The T-DNA region of the plasmid responsible for hairy root in the Agrobacterium rhizogenes strain NCPPB2659 is identified and characterized by its physical map.

Genetic analysis of Tn7 transposition.

The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7. Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites. The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined. All the deleted Tn7s isolated in this work had lost their transposing capability. The possibility of complementing them was studied using plasmids containing all or part of Tn7. Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function. Four other deleted Tn7s could be complemented by Tn7. One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition. The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb. Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7. The classification of Tn7 with regard to the other transposable elements is discussed.

Tn7-encoded proteins.

Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1::Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the left-hand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.

Absence of cis-acting transposition immunity with Tn7.

It is possible in two steps to insert into the plasmid RP4 two copies of the transposon Tn7. This was demonstrated using a wild-type Tn7 in the first step, and a Tn7 derivative (carrying an additional marker), in the second step. The two successive transpositions occurred with the same polarity and frequency. The genetic structures of the resulting plasmids, predicted from the phenotypes of the bacterial host, were confirmed by direct analysis of the plasmid DNAs. Thus, the phenomenon of cis-acting transposition immunity, described with Tn1 or Tn3, does not take place in the case of Tn7.

Restriction map of Tn7.

Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7 delta 6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.