Peter Starlinger

Oo and strong-polar mutations in the gal operon and insertions

SummaryThree λ dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal+ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.

The DNA sequence of the transposable element Ac of Zea mays L.

SummaryThe sequence of the Ac element isolated from the wx-m7 allele has been determined. The Ac element is 4563 bp long. A central portion of roughly 3.1 kb is occupied by three open reading frames, two of which point in one direction and the third in the opposite direction. One of the reading frames potentially encodes a protein with a ten-fold repeat of pro gluN and pro glu dipeptides near its N-terminus. The sequences outside the open reading frames are characterized by the presence of a number of direct and inverted repeats. The Ac element may thus have evolved from a simpler progenitor structure. The sequence we have determined for the Ac from the wx-m7 allele differs in a few key positions from that reported for the Ac element from the wx-m9 allele (Pohlman et al. 1984). We have resequenced these positions in both Ac elements and find them to be identical. We conclude that the phenotypic differences between the two waxy alleles are not caused by structural differences in the Ac elements but rather may be attributable to the differences in their insertion sites.

Structure of the sucrose synthase gene on chromosome 9 of Zea mays L

The structure of the shrunken gene of Zea mays encoding sucrose synthase (EC 2.4.1.13) was determined by (i) sequencing the transcription unit and ˜1.2 kb of 5′ ‐upstream sequences from a genomic clone, (ii) by sequencing a nearly full length cDNA clone and (iii) by determining the transcription start site by a combination of primer extension experiments with synthetic oligodeoxynucleotide primers and S1 mapping. The sucrose synthase gene is 5.4 kb long, of which 2746 bp are found in the mature mRNA. The gene is interrupted by 15 introns. The first two introns are ˜1 kb and ˜0.5 kb in length, respectively, while the other introns are much smaller. A TATA box is located 30 bp upstream from the transcription start site. Approximately 610 bp upstream of the transcription start site a direct repeat of 16 nucleotides, separated by a 4‐fold repetition of the sequence GGTGG is detected. The 16‐bp sequence has similarities to a sequence repeat found between two promoters of a maize zein gene also expressed in the endosperm tissue. The transposable element Ds in the mutant sh‐m5933 and sh‐m6233 alleles is inserted in the seventh and first intron, respectively. The genomic and cDNA clones were obtained from different maize lines. This allows the determination of polymorphic sites which are frequent in 3rd codon position and absent in 1st and 2nd codon positions. In addition, the 3′ ‐untranslated sequence shows two duplications that may have arisen by the insertion and subsequent excision of transposable elements.

Phenotypic assay for excision of the maize controlling element Ac in tobacco.

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac△), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac△ did not form any calli resistant to 200 µg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin‐resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.

IS-Elements in Microorganisms

The bacterial chromosome, while evolving by point mutations in its DNA, is thought to be rather stable with regard to its gross organization. For example, even though the DNAs of Salmonella and Escherichia cross-hybridize only weakly due to divergent sequence evolution (Demerec and New, 1965; Brenner et al., 1969), the genetic maps of the two bacteria are rather similar with respect to the order of functions (Sanderson and Demerec, 1965).

The Shrunken gene on chromosome 9 of Zea mays L is expressed in various plant tissues and encodes an anaerobic protein.

SummaryThe Shrunken gene, located on the short arm of chromosome 9 of Zea mays, encodes the enzyme sucrose synthase (EC 2.4.1.13). The gene is known to be expressed in the endosperm of the developing maize kernel and seems to be involved in sucrose breakdown prior to starch synthesis. We have analyzed different tissues of the maize plant for transcripts of the Shrunken gene and have found rather high transcription rates in the etiolated shoot and the primary root of the germinating kernel. If the etiolated seedlings are illuminated, the transcript level drops by about 95% in the greening plant parts (1st and 2nd leaves) which are active in photosynthesis. A very low transcript level is found in mature green leaves where sucrose is formed from products of photosynthesis via a separate pathway. Upon anaerobic stress of the young seedling, the level of Shrunken transcripts increases 10 and 20 times in shoot and root tissue respectively. Apparently anaerobic induction supersedes the negative control that is observed after illumination in the 1st and 2nd leaves. From the experiments out-lined here we conclude that the anaerobic protein 87 (ANP87, Hake et al. 1985) is encoded by the Shrunken locus.While the expression of the Shrunken gene varies in different tissues and in response to external stimuli, transcription of the second sucrose synthase (B) gene seems to be irresponsive to anaerobic stress and to be expressed at a similar low level in all of the tissues examined.

Transcription of transposable element Activator (Ac) of Zea mays L

Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5‐kb mRNA with an abundance of 1–3 x 107 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5‐kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5′‐end of the transcript was determined by Northern hybridization and S1 mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUG‐free leader 600‐700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3′‐sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the first AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.

Transposition of the maize transposable element Ac in Solanum tuberosum

SummaryThe maize transposable element Ac has been introduced into potato via the T-DNA (transferred DNA) of Agrobacterium tumefaciens. Ac was inserted within the untranslated leader region of a neomycin phosphotransferase II (NPT-II) gene such that excision restored NPT-II activity. Two approaches to monitor Ac excision were used. (i) Using an Agrobacterium strain harbouring plasmid pGV3850::pKU3, leaf discs were selected on kanamycin (Km) after exposure to Agrobacterium. (ii) Using a strain containing plasmid pGV3850HPT::pKU3, the leaf discs were selected on hygromycin (Hm) and the resulting shoots were checked for NPT-II expression. Thirteen kanamycin resistant shoots transformed with pGV3850::pKU3 were isolated, suggesting that Ac had excised from the NPT-II gene. Out of 43 hygromycin resistant shoots transformed with pGV3850HPT::pKU3, 22 expressed the NPT-II gene, indicating that Ac had undergone excision in approximately 50% of the hygromycin resistant shoots. Southern analysis revealed that all kanamycin resistant plants contained the DNA restriction fragments expected when Ac excises from the NPT-II gene. The presence of Ac at new locations within the genomic DNA of several transformants was also detected.

Two kinds of insertions in bacterial genes

SummarySix insertion mutations in the gal operon of E. coli and two insertion mutations in the xycIIOP operon of bacteriophage lambda were tested for homology by annealing separated strands of lambda dgal DNA carrying the insertions, and inspection in the electron microscope.Class 1, consisting of the gal mutations OP 128, OP 141, T-N 116, OP 306, T-N 102 and the lambda mutation r14 are about 800 nucleotide pairs long, completely homologous and not circularly permuted. The first three insertions of class 1 are integrated in one direction with respect to the adjacent genes, the other three in the opposite direction. The DNA inserted in this class of mutations is called IS1.Class 2 consists of the gal insertion OP 308 and the lambda insertion r32. They are about 1400 nucleotide pairs long. The two are integrated in opposite direction with respect to the chromosome of λdgal. The DNA in insertion mutations of class 2 will be called IS 2. IS1 and IS2 do not share any detectable homology.These data are supported by cross-hybridization experiments using RNA transcribed in vitro from lambda dgal or lambda DNA carrying one insertion and DNA carrying either the same or a different insertion.Similar results were obtained by Malamy, Fiandt, Szybalski and Fiandt, Szybalski, Malamy (accompanying papers).

Isolation and molecular analysis of the maize P locus

SummaryThe maize P locus is involved in the synthesis of a red flavonoid pigment in the pericarp, cob and other floral tissues. The tissue-specific pattern of expression of certain P alleles suggests that P may be a complex locus, with more than one functional unit. The P-VV allele, which specifies variegated pericarp and variegated cob, however, shows that insertion and excision of the transposable element Ac affects both pericarp and cob expression as though cob and pericarp pigmentation are controlled by a single gene. Using Ac as a transposon tag, we have isolated 34 kb of genomic DNA from the P-VV and P-RR allele. The cloned DNA contains two 5.8 kb cross-hybridizing regions, in direct orientation relative to each other, separated by 6.6 kb of intervening DNA. A sequence motif of 250 by is repeated at three locations within the cloned region: once within each of the 5.8 kb repeats, and once outside the 5.8 kb repeats. DNA fragments flanking the Ac element detect five transcripts in RNA from wild type (P-RR) that are absent from mutant (P-VV) tissues. To localize the transcribed sequences, DNA probes spanning the 34 kb of cloned DNA were used in Northern analysis of RNA from mutant and wild-type kernels. The results suggest the presence of a single transcriptional unit located primarily within the DNA between the 5.8 kb repeats. The five RNAs transcribed from this region may be formed by alternative splicing. The size of the P gene derived from the length of the transcribed region seems much smaller than the gene size estimated from Ac-induced P-VV mutations.