Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer.

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.

Transfer and Integration of T-DNA without Cell Injury in the Host Plant.

Agrobacterium colonizes plant cells via a gene transfer mechanism that results in plant tumorigenesis. Virulence (vir) genes are transcriptionally activated in the bacteria by plant metabolites released from the wound site. Hence, it is believed that agrobacteria use injuries to facilitate their entrance into the host plant and that the wounded state is required for plant cell competence for Agrobacterium-mediated gene delivery. However, our experiments using vir gene-activated bacteria sprayed onto tobacco plantlets demonstrated that cells in unwounded plants could also be efficiently transformed. The condition of the plant cells was monitored using [beta]-glucuronidase under the control of a wound-inducible promoter. Infection of leaf tissue is light dependent, and it is drastically reduced when abscisic acid is exogenously applied to the plant. Under these experimental conditions, stomatal opening seems to be used by Agrobacterium to circumvent the physical barrier of the cuticle. These results thus show that the proposed cellular responses evoked by wounding in higher plants are not essential for Agrobacterium-mediated transformation.

Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared.

Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro.

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.

Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident.

To evaluate the genetic consequences of radioactive contamination originating from the nuclear reactor accident of Chernobyl on indigenous populations of plants and animals, it is essential to determine the rates of accumulating genetic changes in chronically irradiated populations. An increase in germline mutation rates in humans living close to the Chernobyl Nuclear Power Plant site, and a two- to tenfold increase in germline mutations in barn swallows breeding in Chernobyl have been reported. Little is known, however, about the effects of chronic irradiation on plant genomes. Ionizing radiation causes double-strand breaks in DNA, which are repaired via illegitimate or homologous recombination. We make use of Arabidopsis thaliana plants carrying a beta-glucuronidase marker gene as a recombination substrate to monitor genetic alterations in plant populations, which are caused by nuclear pollution of the environment around Chernobyl. A significant (p<0.05) increase in somatic intrachromosomal recombination frequencies was observed at nuclear pollution levels from 0.1-900 Ci/km2, consistent with an increase in chromosomal aberrations. This bioindicator may serve as a convenient and ethically acceptable alternative to animal systems.

A sensitive transgenic plant system to detect toxic inorganic compounds in the environment.

We describe a transgenic plant-based assay to study the genetic effects of heavy metals. Arabidopsis thaliana plants carrying a beta-glucuronidase (GUS) marker gene either with a point mutation or as a recombination substrate were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants. Transgenic test plants sown on media contaminated by the salts of the heavy metals Cd2+, Pb2+, Ni2+, Zn2+, Cu2+, and As2O3 exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal recombination and point mutation. The test was applied to monitor the genotoxicity of soils sampled in sites contaminated with several heavy metals. Our results indicate that this is a highly sensitive system for monitoring metal contamination in soils and water.

Biomonitoring the genotoxicity of environmental factors with transgenic plants.

All organisms must react to constantly changing surroundings. Environmental factors are thus powerful forces continuously shaping the genomes of all species. Induced genetic changes can be followed using a biomonitor - a living organism that reacts to a given compound in the environment. A vital but challenging task is identifying organisms with which to study the influence of changing environmental conditions. Plants are especially valuable biomonitors. Here, we describe the use of transgenic plant systems to evaluate the genotoxicity of chemical and radiological compounds. We evaluate the potential of further transgene-based systems for studying somatic and germ-line mutations.

Elimination of selection markers from transgenic plants.

Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed.

The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens.

The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells. We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far. In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs. However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence. The importance of the C terminus of VirD2 for T-DNA transfer is discussed.

A small cosmid for efficient cloning of large DNA fragments.

The production and use of the 6 kb cosmid pHC79, a derivative of pBR322, is described. It can be used for cloning of fragments cleaved by EcoRI, ClaI, BamHI (also BglII, BclI, Sau3A and MboI), SalI (also XhoI and AvaI), EcaI and PstI. Hybrid cosmids containing inserts in the size range of 40 kb are packaged in vitro and transduced with an efficiency of 5 X 10(4) - 5 X 10(5) clones/microgram of insert DNA. Prefractionation of the DNA fragments to be cloned into 40 kb sized fragments ensures the cloning of contiguous stretches of DNA. Proteins produced in vitro by the cosmid pHC79 are identical to the ones produced by its pBR322 parent.

Infectivities of native and cloned DNA of cauliflower mosaic virus.

Infectivity assays on turnips reveal that (i) cauliflower mosaic virus (CaMV) DNA, whether circular or linear, is as infectious as the complete virus; (ii) linear DNA obtained with restriction enzymes from the native CaMV DNA has the same specific infectivity as when first cloned in plasmid (pBR322) or bacteriophage (lambda gtWES) vectors and then restricted at the cloning site; (iii) in all cases studied mosaic symptoms are accompanied by virus production. DNA isolated from these viruses is again circular and possesses the three "gaps" characteristic of CaMV DNA. The cloned CaMV DNA, when linked to the vector DNA, is noninfectious or exhibits very low infectivity.

Restriction map of native and cloned cauliflower mosaic virus DNA.

Cloned CaMV DNA replicates faithfully in Escherichia coli, since the restriction map of the cloned DNA can be superimposed over that of the native viral DNA. However, some short fragments were difficult to detect in the restricted native viral DNA, whereas they formed clear bands when derived from cauliflower mosaic virus (CaMV) DNA clones propagated in the E. coli host. Apparently, the small fragments that carry variable-length single-stranded gaps present only in native viral DNA, give rise to diffuse weak bands difficult to recognize in gels. Comparison of maps for several CaMV strains permits evaluation of their possible evolutionary relationship.

Plasmid vectors for positive selection of DNA inserts controlled by the lambda pL promoter, repressor and antitermination function.

Hybrid plasmids consisting of pBR322 or pOP203-3 and the EcoRI-D fragment of lambda DNA kill their bacterial host upon expression of a lambda gene (probably the kil function) located either between or across the SalI sites. The plasmids from surviving hosts are acquired deletions that remove the lambda kil gene or insertions that block the transcription of the kil gene. Some plasmids probably carry point mutations. Based on these findings, we constructed two vector plasmids, pKL1 and pHA10, which can be used for a direct positive selection of cloned fragments. These plasmids are particularly useful for the cloning and selection of N-unresponsive termination signals using BamHI and its isoschizomers. The DNA fragments cloned into these plasmids are under control of the strong pL promoter, which can be regulated by the lambda repressor, and the antitermination activity of the N gene product.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

How does the T-DNA of Agrobacterium tumefaciens find its way into the plant cell nucleus?

Agrobacterium tumefaciens causes the crown gall disease in plants by transferring a piece of DNA, the T-DNA, into the genome of the plant cell. The virulence protein VirD2, tightly linked to the T-DNA, is thought to direct it to the plant cell nucleus and to assist it in integration. The VirD2 protein contains two nuclear localization signals (NLS) which are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster type NLS. The second signal is near the C-terminus and is a bipartite type NLS. The involvement of the C-terminal NLS in the entry of the T-DNA into the plant cell nucleus was directly tested in vivo.

Occult axillary lymph node metastases in breast cancer do matter: results of 10-year survival analysis.

Axillary lymph node status is one of the most powerful prognostic factors for patients with breast cancer and is often critical in stratifying patients into adjuvant treatment regimens. In 203 apparently node-negative cases of breast cancer, a combination of immunohistochemical staining and step-sectioning identified occult metastases in 25% of cases. Ten-year follow-up information is available for these patients. Histologic features of the primary tumor and immunohistochemical staining for estrogen receptor, progesterone receptor, Her-2, and p53 were also evaluated. With multivariate analysis, both occult metastases and higher histologic grade of the primary tumor were independent predictors of disease-free survival. Histologic grade was the only significant independent predictor of overall survival. Estrogen receptor, progesterone receptor, Her-2, and p53 status did not predict the presence of metastases or survival when all tumor types were considered together. Metastases >0.5 mm significantly predicted a poorer disease-free survival when invasive ductal carcinomas were considered alone. Histologic grade was significantly associated with disease-free survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients. The presence of occult metastases approached significance for overall survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients.

Risk factors in alcohol associated breast cancer: alcohol dehydrogenase polymorphism and estrogens.

Chronic alcohol consumption is associated with an increased risk for breast cancer, even if consumed in moderate doses. Since acetaldehyde is a carcinogenic factor associated with chronic alcohol consumption, individuals with the alcohol dehydrogenase 1C*1 allele (ADH1C*1 allele) seem to be at particular risk, since this allele encodes for a rapidly ethanol metabolizing enzyme leading to increased acetaldehyde levels. Since recent epidemiological studies demonstrated an increased risk for breast cancer for individuals with the ADH1C*1 allele, we have investigated here ADH1C genotypes in moderate alcohol consumers. Furthermore, estradiols are also known risk factors for breast cancer and acute alcohol ingestion in high doses results in increased serum estradiol concentrations. Thus, in the present study, we tested the effect of low ethanol doses on estrogen serum concentrations. We analyzed the ADH1C genotype in 117 moderate alcohol consumers with breast cancer and in 111 age-matched women with alcohol associated diseases without cancer (74 cirrhotics, 22 patients with pancreatitis and 15 alcohol dependent patients). In addition, 107 healthy controls were studied. Genotyping of the ADH1C-locus was performed using polymerase chain reaction-based restriction fragment length polymorphism methods on leukocyte DNA. To study the effects of ethanol on estradiol levels, ethanol in a dose of 0.225 g/kg body weight was given orally to 8 premenopausal women at various time points of their menstrual cycle. Thereafter estradiol serum concentrations were measured over time. The allele frequency of the ADH1C*1 allele was found to be significantly increased in moderate alcohol consumers with breast cancer as compared to age-matched alcoholic controls without cancer (62% vs. 41.9%, p=0.0035). Women with the ADH1C*1,1 genotype were found to be 1.8 times more at risk for breast cancer than those with another genotype (95% CI 1.431-2.330, p<0.001). Oral ethanol increased serum estradiol levels significantly by 27-38%. The data demonstrate that moderate alcohol consumers with the ADH1C*1 allele have an increased risk to develop breast cancer and even small amounts of alcohol increase serum estradiol levels significantly in premenopausal women especially in the midphase of the menstrual cycle.

Prognostic significance of MUC1 epithelial mucin expression in breast cancer.

The epithelial mucin produced by the MUC1 gene is present in the apical cell membrane of normal breast epithelial cells and is highly expressed in many breast cancers. Several studies have provided conflicting evidence regarding the relationship between MUC1 expression and survival in breast cancer patients. In this study a detailed immunohistological analysis of MUC1 expression was performed using monoclonal antibody BC2 and was related to other tumor characteristics and patient survival. Patients whose tumors showed MUC1 expression in greater than 75% of tumor cells had significantly poorer disease-free and overall survival (P < .05). The proportion of cells showing cytoplasmic MUC1 expression was prognostically significant, but the proportion of cells that lined gland spaces showing apical membrane staining was of no prognostic significance. A high level of MUC1 expression was significantly associated with the presence of axillary node metastases and estrogen receptors but not with other tumor characteristics.

Expression of MUC2 epithelial mucin in breast carcinoma.

AIMS: To examine the expression of the MUC2 epithelial mucin in breast carcinoma; to relate this to patient survival. METHODS: Sections from 210 breast carcinomas were stained with the anti-MUC2 core protein monoclonal antibody, 4F1, using an immunoperoxidase technique. The proportion of tumour cells positively stained and the localisation and intensity of any staining were recorded. Expression of MUC2 was compared with histological type and grade, tumour size, presence of nodal metastases, presence of oestrogen receptors, and menopausal status. The prognostic value of MUC2 expression was examined using Kaplan-Meier survival analysis. RESULTS: MUC2 mucin was detected in 19% of cases of invasive carcinoma, in 11% of cases of carcinoma in situ, where present, but very rarely in adjacent normal breast epithelium. Presence of MUC2 was significantly associated with a shorter disease free interval (p < 0.05), although the observed difference in duration of overall survival was not significant. CONCLUSIONS: The MUC2 detected in breast carcinoma may be underglycosylated or staining may represent detection of the protein core before the completion of glycosylation. The virtual absence of 4F1 reactivity in normal breast epithelium suggests that, unlike the MUC1 mucin, the MUC2 mucin is not highly expressed by these cells. The mechanism by which expression of MUC2 affects the biology of breast tumours is unclear, although expression may be a reflection of general derepression of genes during tumour progression.

T-DNA transfer to maize plants.

Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.