Enhancer transposable element induced changes at the A locus in maize: The a-m-1 6078 allele

Summary Transposable (controlling) elements in maize are highly variable in their versatility in inducing changes at any locus. With the Enhancer (En) (Suppressor-Mutator-Spm) controlling-element system,a multitude of changes result from the action of En on specific alleles. One such allele, McClintock's a-m-1 6078 allele, illustrates the diversity of events that can take place that involve three parameters; namely, spotting-timing, spotting-frequency, and background pigment formation with and without En. A series of derivative alleles has been isolated and described according to the three paramters. The multitude of changes that has been isolated illustrates the striking versatility of these transposing elements to initiate changes at the controlled locus.Journal Paper No. J-11135 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

Basis for the diversity of states of controlling elements in maize

Summary In maize the insertion of a controlling element such as the regulatory element, En, (Enhancer) at a locus generates diverse derivatives. These derivatives include stable phenotypes that range from colorless to full color. Distinguishable patterns are determined by timing and frequency of mutation events. It has been hypothesized that diverse expressions are based either on position of the controlling element at the locus or composition differences among controlling elements. — Experiments with two target loci, A2 and C, indicate that the previous pattern of a controlling element does not influence the pattern generated at a new site. This finding supports the hypothesis that these diverse trans-effects of En are not caused by a change in the genetic information of En itself but rather by its position; diverse expressions are a manifestation of the effect of an element at a specific locus site. — Controlling elements are discussed in relation to chromosome structure, gene activation, gene programming and possible parallels in prokaryotes.Journal Paper No. J-8416 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011. Project No. 1884. Support from NSF GB38328     

Genetic and molecular analysis of a three-component transposable-element system in maize

Summary Two different factors control the mutability of an unstable allele (c2-m8810581) of the C2 gene of maize. Both an autonomous En/Spm element and an unrelated independent factor, named Mediator, are coordinately required for the excision of the insert in c2-m881058Y. According to genetic analysis, Mediator does not have the suppressor (S) function or mutator (M) function of En/Spm. Mediator has no effect on the timing or frequency of excision of Enl, En-low, or various I/dSpm elements. Hence, Mediator only mediates a specific interaction between En and the insert at c2m881058Y. Molecular analysis of c2-m881058Y has revealed a 3.3 kb, complex, En-related receptor element inserted into the second exon of the C2 gene. The ends of this element are homologous to the ends of En/Spm, but an internal l.7 kb region shows no En/Spm homology. A great degree (11–14%) of nucleotide changes, relative to Enl, occur within and between the 12 bp TNPA binding motifs. Alterations of these critical cis-determinants may account for the need for a helper factor for excision. This element is named Irma, for Inhibitor that requires Mediator also, and represents a unique, low copy number class of receptor element.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

The Fcu controlling-element system in maize

Summary The relationship between the Fcu controlling-element system and the spotted-dilute R system was investigated. The Fcu controlling-element system consists of the receptor element allele r-cu and the regulatory element Fcu. The equivalent components of the spotted-dilute R system are respectively R-r#2 (or R-r#2 Dil) and Spf. The R-r#2 allele of the latter system was shown to be responsive (mutable) to Fcu, provided that it has had an uninterrupted association with Dil or Dpf as evidenced by the color variegation of the aleurone tissue. The reverse test, in which the r-cu allele of the Fcu controlling element system was tested for its response to Spf, proved negative. This was surprising in view of the relationship and specificity between systems. The possibility was considered that maize controlling elements may have different sizes as is known for bacterial insertion sequences. —The variable dilute pigmenting capacity of the r-cu allele also was studied. A given level of r-cu-induced pigmentation, despite the wide range in pigmentation expression, was found to be generally non-heritable, as based on a test of correspondence between parent and progeny.Journal Paper No. J-9204 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1884Now with Funk's Seeds, Casalmorano (Cremona), Italy     

An altered state of a specific en regulatory element induced in a maize tiller

There are numerous states of the regulatory element, Enhancer (En). With specific receptor alleles, such as a2m(r-pa-pu) or a2m(r), specific mutability patterns are expressed. One specific derivative En allele, En-v (En-variable), was originally identified with a coarse pattern of mutability with the a2m(r-pa-pu) allele and giving progeny with varied En expression (standard to reduced within an ear progeny). Derivatives of En-v were subsequently found on numerous occasions to give only a very reduced expression (fewer mutant spots) with the a2m(r-pa-pu) allele in the ears derived from the main stalk of the corn plant. When a comparison is made of the effect of this changed En-v state between tiller ears and main stalk ears of the same plant, the tiller ears show an increased level of En-v expression (coarse pattern), while the main-stalk ears continue to show the very reduced level of En-v expression (low frequency of very late variegation). This increased level of mutability of the tiller ears is maintained when transmitted through the main-stalk ear in the subsequent generation. These results indicate that heritable alterations of controlling elements can be produced by endogenous environmental factors present during normal plant development.     

The inveterate wanderer: study of Enhancer wandering on chromosome 3 in maize

The transposition of the maize transposable element Enhancer (En) had been focused on one chromosome 3 for several generations. From the a1-m(Au) allele with an autonomous En, a new En reporter allele a1-m(r)3927-1, was isolated that undergoes very infrequent and late excision events, producing one or two small spots in the aleurone. This allele is seriously impaired in its capacity to excise. Coincident with the origin of this allele, an En was located at a site close to the a1 locus. From this initial insertion site, the movement of this En was followed for three to four generations in 974 families with a higher transposition rate of this En (50% of the testcross progeny) than that found in a previous study of En transposition. This is the first case reported where a particular En was followed for more than three generations. The higher rate of wanderings of this En along the same chromosome led to the term vagabond En (En ^vag ). Genetic evidence that En may transpose from a replicated donor site to an unreplicated site is provided. Speculative mechanisms on the origin of a1-m(r)3927-1 and En ^vag are discussed.Journal Paper No. J-15864 of Iowa Agricultural and Iowa Economical Experiment Station Project #3176     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Change in State following Transposition of a Regulatory Element of the Enhancer System in Maize

From an original A2 allele (colored aleurone), a mutable allele, a2-m-4 1629, that changes from a2 to A2 is described. Mutability is expressed as a very distinct pattern limited to the last cell division.—The mutability of a2-m-4 1629 is autonomously controlled by an En at the a2 locus. This En, inactive on standard a testers for En, is partially active on a2-m-1, an a2 tester for En, and expresses varied levels of activity from limited to nearly full suppression of the a2-m-1 color phenotype.—When the En of the a2-m-4 1629 allele transposes from the a2 locus, it behaves, at the new position, like a standard En in triggering a2-m-1, a-m-1 and a-m(r), which express colored spots on a colorless background. The activity of En is therefore different following the change in chromosome location. This finding supports the "position" hypothesis that has been proposed to explain diverse patterns observed among controlling elements. In this case mutation is related to the terminal cell state and not to tissue differences as shown with some phase-variation regulatory elements.     

Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Chromosome labeling with transposable elements in maize

Transposable elements randomly insert into a targeted locus at a frequency of 10^-6 to 10^-5. The En element has been shown in previous studies to transpose more frequently into closely linked sites. Thus, it is appropriate to place an En element onto each of the 20 chromosome arms in maize to maximize tagging efficiency. This is called chromosome labeling for tagging purposes with transposons. After a chromosome arm has been labeled with a transposon, genes residing in that arm will have a greater chance to be tagged by the transposon. To date, all of the maize chromosome arms have been labeled with at least one of five Encontaining alleles. The elements were linked to the arms using reciprocal translocations. The usage of these arm-labeled lines is discussed in the context of gene tagging.Journal Paper No. 15224 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa; Project No. 3176     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5-CACTA--- -3; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system

Summary In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants —defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements —the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.     

DNA sequence alteration resulting from a mutation impairing promoter function in the lac repressor gene

Summary A deletion of four base pairs in the — 10 homology region of the lacI promoter severely damages I gene expression.     

Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

Summary We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42° C (relative to 30° C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30° C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42° C; and 4) that defects in excision repair or in other Uvr⁺-dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions.     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the “endotoxic principle” of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg²⁺] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70°C. These data were correlated with measurements of the β→α phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges—in the absence of Mg²⁺—from 45°C at high to 56°C at low water content, and—at an equimolar content of Mg²⁺—from 52°C at high to 59°C at low water concentrations. In the gel phase—in which the lipid A acyl chains are more disordered than those from saturated phospholipids—cubic phases are adopted at high water content (>60%) and at high [lipid A):[Mg²⁺] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal H_II, state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg²⁺ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.