Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco.

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

Sequence of three bronze alleles of maize and correlation with the genetic fine structure.

The genomic sequences of three bronze alleles from Zea mays, Bz-McC, Bz-W22 and bz-R, are presented together with their flanking sequences. The bronze locus encodes UDPglucose flavonoid glucosyl-transferase (UFGT), an anthocyanin biosynthetic enzyme. The wild-type alleles Bz-McC and Bz-W22 condition purple phenotypes in the seed and plant, while bz-R conditions a bronze color. A full length cDNA corresponding to the Bz-McC allele was cloned and sequenced. Primer extension and RNase protection experiments were used to verify the 5' end of the bronze transcript. The Bz-McC allele has a 1416-bp coding region, a 100-bp intron and an approximately 83-bp 5' leader. Upstream of the message initiation site the sequences CTAACT and AATAAA occupy the positions where the eukaryotic consensus CCAAT and TATA boxes are normally found. The alleles Bz-McC and bz-R each have different large insertions with characteristics of transposable elements in their 5' flanking regions. The bz-R allele is distinguished by a 340-bp deletion starting within the intron and including 285 bp of the second exon. The Bz-McC and Bz-W22 isoalleles are known to differ in two genetically defined locations. The uts and uqv sites from the Bz-McC allele condition, respectively, lowered thermostability for the UFGT enzyme and increased amount of UFGT activity when compared with the corresponding sites in the Bz-W22 allele. The uts site maps to a region of the gene encoding two adjacent amino acid differences, either or both of which might alter the thermostability of the UFGT enzyme. The difference in UFGT levels conditioned by the uqv site is shown here to be correlated with variation in the bronze mRNA level. A likely cause of this decreased bronze mRNA level in Bz-W22 is a 6-bp duplication near the sequence CTAACT located 74 bp upstream of the bronze message initiation site. This region is therefore tentatively identified as the uqv site.

The frequency of transposition of the maize element Activator is not affected by an adjacent deletion.

The maize mutable allele bz-m2 (Ac), which arose from insertion of the 4.6 kb Ac element in the bz (bronze) locus, gives rise to stable bz (bz-s) derivatives that retain an active Ac element closely linked to bz. In the derivative bz-s: 2114 (Ac), the Ac element is recombinationally inseparable from bz and transposes to unlinked sites at a frequency similar to that in the progenitor allele bz-m2 (Ac). Both alleles have been cloned and sequenced. The bz-s: 2114 (Ac) mutation retains Ac at the original site of insertion, but has lost a 789 bp upstream bz sequence adjacent to the insertion, hence the stable phenotype. The 8 bp target site direct repeat flanking the Ac insertion in the bz-m2 (Ac) allele is deleted in bz-s: 2114 (Ac), yet the Ac element is not impaired in its ability to transpose. The only functional Ac element in bz-s: 2114 (Ac) is the one at the bz locus: in second-cycle derivatives without Ac activity, the loss of Ac activity correlated with the physical loss of the Ac element from the bz locus. The deletion endpoint in bz-s: 2114 (Ac) corresponds exactly with the site of insertion of a Ds element in a different bz mutation, which suggests that there may be preferred integration sites in the genome and that the deletion originated as the consequence of an abortive transposition event. Finally, we report two errors in the published Ac sequence.

Stability of deletion, insertion and point mutations at the bronze locus in maize.

Phenotypic revertants from several kinds of mutations, including deletions, have been detected by pollen analysis at the wx and Adh loci in maize. Mutations in these genes give phenotypic revertants with median frequencies of 0.7 and 0.5×10(-5), respectively. However, the nature of such revertants can only be analyzed following their recovery from conventional matings. In the current study large seed populations derived from crosses involving several bz (bronze) locus mutations in maize were examined for reversion to a Bz (purple) expression. Deletion, insertion and point mutations were included in the study. Principally, over 2 million gametes of the bz-R mutation, which is shown here to be associated with a 340 base pair deletion within the transcribed region of the gene, have been screened for reversion. No revertants from it or any of the other bz mutations have been recovered, even though a total of almost 5 million gametes from homoallelic crosses have been examined to date. Results from seed analysis are discussed in reference to those from pollen analysis in maize.

Bacteriophage SPO1 structure and morphogenesis. II. Head structure and DNA size.

The capsid of bacteriophage SPO1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number T = 16. The major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor. Tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends. The lattice constant of these structures is 13.4 nm; diameter is 109.5 nm. The size of the double-stranded SPO1 DNA (containing 5' hydroxymethyl uracil in place of thymine) was measured by sedimentation analysis and electron microscopy and has a molecular weight of 86 X 10(6) (about 140 kilobase pairs), which is smaller than several previously reported values.

Dinitrogen fixation in male-sterile soybeans.

Partial male-sterile (ms(4)/ms(4)) soybeans (Glycine max L. Merr.) and their fertile isoline (Ms(4)/Ms(4)) were grown in adjoining field plots. From 62 until 92 days after emergence, the nitrogenase activity, assayed by acetylene reduction, of the average male-sterile plant was approximately twice that of the average fertile plant. At approximately 100 days after emergence, the assayable nitrogenase activity of the fertile plants fell to zero, whereas the nitrogenase of the partial male-sterile plants continued to be active for two additional weeks. Thus, this male-sterile plant seems to fix dinitrogen both at a higher rate and over a longer duration than does its fertile isoline.

Hydroponic growth and the nondestructive assay for dinitrogen fixation.

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK(a) 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.

Rotor speed dependent sedimentation of circular and linear DNA.

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.

Rotor speed dependent sedimentation of circular and linear DNA.

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.