Biomedical subjects

M A Matzke

Publications and source records attributed to M A Matzke.

44 records · Page 3Linked to original sources

PubMed1991

Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences.

In a previous study on doubly transformed tobacco plants, we observed the unexpected inactivation in trans of T-DNA-I (encoding KanrNOS) following the introduction into the same genome of an unlinked copy of T-DNA-II (encoding HygrOCS). This inactivation, which probably resulted from interactions between homologous regions on each T-DNA, was correlated with methylation in the nospro, which controlled the expression of both the nptII and nos genes. In this paper, we show that the inactivation and methylation of the nos(pro)nptII gene in the presence of a suppressor T-DNA-II locus can be either complete (epistasis) or partial (cellular mosaicism). In plants showing partial suppression, the strength of the Kanr phenotype, which apparently reflected the proportion of cells expressing the nptII gene, was inversely correlated with the degree of methylation of the nos(pro). The extent of nos(pro) methylation decreased progressively in successive generations as suppressor T-DNA-II loci were crossed out. The strength of the Kanr phenotype was improved and nos(pro) methylation was less extensive in first generation Kanr progeny obtained from outcrossing with untransformed tobacco than from self-fertilization.

Amino Acid Oxidoreductases↗

PubMed1990

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.

Animals↗

PubMed1990

Deletion analysis of a zein gene promoter in transgenic tobacco plants.

A zein gene (Z4) promoter containing 886 bp upstream from the transcription start site has been shown previously to be active specifically in the endosperm of transgenic tobacco seeds. To investigate the region required for this tissue-specific activity, deletions of the Z4 promoter were constructed and placed upstream of the beta-glucuronidase (GUS) reporter gene. When these deletions were tested in transgenic tobacco plants, seed-specific GUS activity, which reached a peak between 15 and 19 DAP, was observed for promoters extending from -886 to -174. Interestingly, the 174 bp promoter lacked the complete 15 bp consensus sequence found in the same position in all zein genes so far sequenced. With the next shorter promoter in the deletion series (79 bp), which just included the CAAT and TATA elements, negligible GUS activity was observed in seeds. The results demonstrated that 174 bp upstream of the transcription start site are sufficient for tissue-specific and temporally regulated activity of the Z4 promoter in tobacco. At most, two-fold enhanced activity was observed with additional 5' sequences up to -886.

Base Sequence↗

PubMed1990

A 41-42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes.

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41-42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.

Animals↗

PubMed1989

Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants.

Doubly transformed tobacco plants were obtained following sequential transformation steps using two T-DNAs encoding different selection and screening markers: T-DNA-I encoded kanamycin resistance and nopaline synthase; T-DNA-II encoded hygromycin resistance and octopine synthase. A genetic analysis of the inheritance of the selection and screening marker genes in progeny of the doubly tranformed plants revealed that the expression of T-DNA-I genes was often suppressed. This suppression could be correlated with methylation in the promoters of these genes. Surprisingly, both the methylation and inactivation of T-DNA-I genes occurred only in plants containing both T-DNAs: when self-fertilization or backcrossing produced progeny containing only T-DNA-I, expression of the genes on this T-DNA was restored and the corresponding promoters were partially or completely demethylated. These results indicated that the presence of one T-DNA could affect the state of methylation and expression of genes on a second, unlinked T-DNA in the same genome.

Journal Article↗

PubMed1988

Endosperm-specific activity of a zein gene promoter in transgenic tobacco plants.

Transgenic tobacco plants containing maize gene (Z4) encoding a 23-kd zein protein, which is normally synthesized in the endosperm of maize seeds, were obtained using a modified Ti plasmid vector. Although a polyadenylated transcript homologous to the Z4 gene was present in the seeds of some of these transgenic plants, zein protein could not be detected in any of the plants tested (35 total). To simplify the analysis of the tissue specificity of the Z4 promoter (Z4(pro)) in different organs of transformed tobacco plants, additional transgenic plants containing the chimeric genes Z4(pro)-CAT and Z4(pro)-GUS were produced. Very weak seed-specific CAT activity was observed in one out of ten Z4(pro)-CAT-transformed plants. When the more sensitive GUS assay system was used to evaluate Z4(pro) activity in tobacco, it could be shown in all 11 transgenic plants obtained that GUS activity was restricted to the endosperm tissue of transgenic tobacco seeds. To study the synthesis and stability of the zein proteins in different organs of transgenic tobacco plants, the Z4 protein-coding region and also a cDNA clone (A30) encoding a 19-kd zein protein were placed under the control of the 35S promoter (35S(pro)) of cauliflower mosaic virus. Undegraded zein proteins of both size classes were detected in roots, leaves and endosperm tissue, but not embryos, of mature seeds from 35S(pro)-zein-transformed plants. The zein proteins did not appear to be broken down during tobacco seed germination; synthesis of zeins began in green cotyledons.

Journal Article↗

PubMed1986

Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization.

We have used in situ hybridization to determine the sites of insertion of Agrobacterium rhizogenes Ri T-DNA in the chromosomes of Crepis capillaris (2n = 6) transformed roots. Four transformed root lines were obtained by infecting Crepis stem segments with A. rhizogenes. Southern hybridization analysis indicated that each root line was the result of one or more independent T-DNA insertion events. In two root lines, one copy of T-DNA was present; the other two root lines each contained two copies of T-DNA. To localize these T-DNA inserts on Crepis chromosomes, metaphase spreads were perpared from each root line, and hybridized in situ to a biotinlabeled T-DNA probe. The results indicated that T-DNA was present in a different chromosomal location in each root line, and that each chromosome had been a target for T-DNA insertion at least once. In the root lines containing two T-DNA inserts, two patterns of integration were observed: in one case the T-DNAs were present on separate chromosomes; in the other case the two T-DNAs were close together (but not tandemly arranged) on a single chromosome. A comparison of these results and those obtained previously for a fifth Crepis-transformed root line demostrated that Ri T-DNA does not insert preferentially into a particlar chromosomal location.

Journal Article↗

PubMed1984

Transcription of a zein gene introduced into sunflower using a Ti plasmid vector.

A maize genomic clone containing a zein gene (Z4) was inserted into the T-region of the T37 Ti plasmid. Agrobacterium tumefaciens cells carrying this modified Ti plasmid were used to inoculate sunflower stemlets. Callus tissue active in nopaline synthesis was grown from a single transformed cell. DNA analysis of this tissue showed that the zein gene plus T-DNA was present in approximately 12 copies per diploid sunflower genome. A 1000 +/- 100 base RNA homologous to a zein probe could be isolated from the engineered sunflower tissue and the 5' end of this RNA was determined by S1 nuclease mapping. Two transcription start sites were detected. The positions of these transcription start sites and the ratio of the amounts of the two transcripts are identical for the Z4 gene in sunflower and in maize endosperm. Although the zein RNA isolated from the engineered sunflower tissue could be translated in a wheat germ system to yield an immuno-precipitable protein of the expected mol. wt., the presence of the zein protein in the sunflower tissue could not be demonstrated.

DNA, Recombinant↗