SIZE: a program to determine the size of nucleic acid and protein molecules from gel mobilities.

The relationship between fragment length and the reciprocal of mobility and the least-squares curve fitting analysis for this relationship have been combined into a user-friendly program for determining the size of unknown DNA fragments by reference to known standards. Protein sizes can be determined in the same way. The program offers an accurate, simple-to-use mechanism for sizing molecules that requires only an IBM-compatible computer.

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells.

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5' flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells.

CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.

Somaclonal variation in a maize inbred line is not associated with changes in the number or location of Ac-homologous sequences.

Somaclonal variation (tissue culture-induced mutations) may result, in some instances, from the activation of transposable elements. This study was conducted to determine whether somaclonal variants in the Zea maize L. inbred line FR27rhm were associated with movement of the transposable element Activator (Ac). Ten variants, seven of which from genetic analyses fit a single recessive gene model and three which did not due to a low number of mutant plants, were selected for analysis. Total DNA from these and from uncultured FR27rhm seedlings were examined by Southern blot analysis using the internal 1.6-kb Hind III fragment derived from the cloned Ac7 element as a probe. By using a restriction endonuclease which does not cut within the element, the number and distribution of the copies of Acrelated sequences in the FR27rhm genome could be determined. From the number of bands seen in the blots, we conclude that the FR27rhm inbred contains large numbers of Ac -related sequences. However, the pattern of bands seen in the ten variants and in the uncultured seedlings were identical, indicating that there had been no movement of any of the Acrelated sequences to cause the tissue culture-induced mutations.

Restriction fragment length polymorphism (RFLP) of wild perennial relatives of soybean.

Total DNA from callus tissue of 28 accessions representing seven wild perennial Glycine species was compared using recombinant genomic probes derived from G. max, the soybean. Using two probes, we show that this molecular approach both confirms and extends the model for the taxonomic relationships between the species derived from morphological and cytogenetic data, and that it provides clear evidence that RFLP analysis of genomic sequences has the potential for revealing the derivation of the member species of the wild perennial Glycine taxon. Although, in this preliminary report, the sample size for each species is small, it is clear that the greatest between-accession variation occurs in G. tabacina (B2B2) and G. clandestine (A1A1), suggesting that these may be the taxa from which further speciation occurred in the subgenus.

Right-border sequences enable the left border of an Agrobacterium tumefaciens nopaline Ti-plasmid to produce single-stranded DNA.

The T-region of nopaline-type Ti-plasmids (the portion of the plasmid that is transferred to plant cells) of Agrobacterium tumefaciens is delimited by 23-25 bp direct repeats. They are nicked by the products of the virD locus and the presence of these nicked sites is correlated with the synthesis of single-stranded T-region copies. Despite previous indications to the contrary, we show that the pTiT37 T-region left border is capable of producing single-stranded DNA with high efficiency and that its ability to do so is totally dependent on right border-proximal cis-acting sequences, most probably overdrive, located several kilobases from the border. The absence of overdrive does not affect the single-strand nicking activity of the virD product but only the production of single-stranded copies from the nicked substrate.

Ribosomal DNA methylation in a flax genotroph and a crown gall tumour.

The methylation patterns of two flax lines are described. One, a genotroph S1, has 800 rNA genes per haploid cell while FT37/1, a crown gall tumour incited on S1, has only 300. Using the enzymes EcoRII, BstNI and ApyI to assess CXG methylation and HpaII and MspI for CG, we show that the methylation patterns of the rDNAs of both lines are identical. Both lines contain 3 fractions; the first contains repeats that are methylated at all sites examined and the second has some unmethylated sites. The third fraction contains repeats that are fully methylated but contain a discrete hypomethylated site at the 5' end of the pre-rRNA. The number of repeats which show these hypomethylated sites is constant in both lines despite the copy number difference. These may represent the active rRNA gene repeats.

The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids.

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.

The effect of right terminal repeat deletion on the oncogenicity of the T-Region of pTiT37.

A modified pTiT37 plasmid was constructed by deleting a 103 base fragment between an AhaIII and a Bc/I site. This fragment, located to the right of the nopaline synthase gene contains the right terminal 25 base pair repeat sequence which defines the right limit of the T-Region. The effect of this deletion was determined on a number of host plants. In contrast to previous reports, the deletion does not destroy tumorigenicity on all plant species. It had no effect on tumorigenicity when Linum usitatissimum was used as the test species and an attenuating effect when Kalanchoë tubiflora was used. Only when Nicotiana tabacum was used did the mutant appear avirulent. We propose from these data and the phenotype of those tumours that form, that a pseudo border located in the 3' untranslated region of the ipt locus has been used to provide the right hand limit of the T-Region in the absence of the normal border.

Extra DNA in the T region of crown gall Ti-plasmid pTiA66.

The mutant tumorigenic phenotype of pTiA66, a derivative of the broad host range octopine Ti-plasmid pTiA6, results from a 2.6-Kb insertion into EcoRI fragment 32g of the T region, which has been implicated in the auxin synthesis disruption tumor character. The inserted DNA is closely related to sequences from BamHI fragment 11 of the same or a related plasmid but probably originally derives from a chromosomal sequence.

The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor.

The FT37/1 tumor line induced by Agrobacterium tumefaciens on flax epicotyls contains 22-24 copies of the T-DNA encoded nopaline synthase gene per cell. All the gene copies are methylated to some extent but the methylation is not uniform, nor does it reflect the methylation level of the flanking plant DNA. This extensive methylation correlates with an extremely low level of expression of the nopaline synthase gene. Treatment of the tumor line with the in vivo demethylating drug 5-azacytidine at a concentration of 3 X 10(-5) M, resulted in the demethylation of, on average, one copy of the nopaline synthase gene per cell. This demethylation was paralleled by an increase in the transcription of the gene and indicates that cytosine methylation is capable of suppressing the expression of plant genes in vivo.

Nopaline Ti-plasmid, pTiT37, T-DNA insertions into a flax genome.

The FT37/1 plant tumor line induced on flax epicotyls by T37, a nopaline strain of Agrobacterium tumefaciens, contains multiple copies of the pTiT37, T-DNA. There are three to four distinct full-length insertions and one tandem insertion. Allowing for the different copy numbers of the inserts, this amounts to seven to eight T-DNA copies per genome unit. The genome unit in this case is the haploid DNA value (7 X 10(8) bp) which predicts a T-DNA copy number of 14-16 per diploid cell. Three novel types of abnormal insertion are also present. FIL (one copy per basic genome) comprises 3.04-4.47 kb of T-DNA derived from the left end, with a normal left border and an abnormal right border. FIR (four copies per basic genome) comprises 5.88-6.47 kb of T-DNA derived from the right end, with a normal right border and an abnormal left border. The third abnormality is represented by fragment "X," a HindIII fragment of 4.90 kb which contains homology with several noncontiguous regions of the T-DNA and which may derive from a tandem insertion. Of the two possible left-border sites (primary and secondary) in which fusion with plant DNA sequences has been observed, only the primary is used.

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.

Sequence organization of the soybean genome.

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.

Molecular integrity of plant ribosomal ribonucleic Acid.

Thermal denaturation of plant ribosomal RNA followed by gel fractionation shows that although a large percentage of molecules contain breaks in the polynucleotide chain, 25S and 18S RNAs do exist as unique molecular species. Values for the rate constant of hydrolysis under routine denaturing conditions are of the order of 10(-7) to 10(-8) sec(- 1) and these are shown not to be a result of ribonuclease activity. This high rate of hydrolysis and the use of insensitive fractionation procedures may account for the reported absence of a 25S rRNA molecule and its apparent conversion to a molecule similar in size to 18S RNA.