Mol Gen Genet (1987) 208:107-113 © Springer-Verlag 1987 Characterization of a null-allele for the Gy 4 glycinin gene from soybean* Bernard J. Scallon, Craig D. Dickinson, and Niels C. Nielsen USDA/ARS and the Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA Summary. A null allele for the Gy4 glycinin gene from the Materials and methods cultivar Raiden was sequenced and compared with a func- tional Gy4 gene from another cultivar. The results showed Materials. Restriction enzymes were purchased from either that the null phenotype probably resulted from a point mu- Bethesda Research Laboratories (BRL) or Boehringer tation that changed the translation initiation codon from Mannheim. T4 ligase was from New England Biolabs. T4 ATG to ATA. Transcripts of the mutant gene were detected polynucleotide kinase and nick translation kits were from in total RNA from seed, but the mRNAs did not become BRL. The c~-[32p]dNTPs were from Amersham and 7- associated with polysomes as did functional Gy4 message [32p]ATP was from ICN Nutritional Biochemicals Inc. Ni- in the control cultivar. This was probably due to premature trocellulose was purchased from Schleicher and Schuell. Ri- dissociation of mutant gY4 mRNA from ribosomes due to bonuclease A (RNAase A), ribonuclease T1 (RNAase T1) nonsense codons during translation of an incorrect reading sucrose and Ficoll were from Sigma Chemical Company. frame. NZY broth was purchased from Gibco Laboratories. Lambda DNA packaging extracts, DNase I and RNasin Key words: Glycinin mutant - Storage protein - DNA se- were purchased from Promega Biotec. Charon 35 bacterio- quence phage were kindly provided by Dr. Jim Mullins, Harvard School of Public Health, Boston, Mass. Isolation ofleafDNA. Plants of the soybean cultivar Raiden Introduction were grown in the greenhouse and leaves harvested when Investigations into the molecular basis of null alleles afford one or two trifoliate leaves had fully emerged. The leaves an opportunity to learn what types of genetic lesions can were immediately frozen in liquid nitrogen and then stored affect gene expression. Although a number of null alleles at -80 ° C until used. DNA was isolated from the leaves have been identified, only a few have been characterized by the mini-preparation method of Dellaporta et al. (1983) at the molecular level. These include null alleles for maize with two modifications: (l) the leaves were not lyophilized alcohol dehydrogenase (Bennetzen et al. 1984; Taylor and prior to grinding, and (2) following precipitation with cethyl Walbot J985), soybean seed lectin (Goldberg et al. 1983), trimethylammonium bromide, the leaf DNA was treated and the 0(-subunit of fl-conglycinin (Ladin et al. 1984). The with 30 gg/ml of RNAase A for 30 min at 37 ° C. The DNA mutations in these alleles are due to insertions or deletions was extracted once with phenol/chloroform and once with that affected transcription. chloroform before precipitating with ethanol. Null alleles have also been reported among members of the gene family that encode glycinin subunits from soy- Construction and screening of a genornic library. Raiden leaf beans. The best characterized of the glycinin null pheno- DNA was partially digested with MboI and then size-frac- types is found in the cultivar Raiden. It lacks the AsAgB3 tionated on a 5%-20% NaC1 gradient. Fractions contain- subunit which is encoded by the Gy4 gene (Staswick and ing DNA fragments between 14 and 21 kb in size were Nielsen 1983; Kitamura etal. 1984, 1980). The purpose pooled and the DNA was precipitated with ethanol. Charon of the work described in this communication was to identify 35 arms (Loenon and Blattner 1983) were prepared by a the genetic lesion responsible for the absence of the AsA4B3 modification of a method described by Maniatis et al. subunit in seeds in the cultivar Raiden. (1982). The cos sites of Charon 35 DNA were annealed for 1 h at 42 ° C in 100 mM Tris-HC1, pH 8.0 and 10 mM * Cooperative research between USDA/ARS and the Indiana Agri- cultural Experiment Station. This is Journal Paper No. 10,884 from MgC12 (80-120 gg of DNA in 400 lal volume). The anneal- the Purdue Agricultural Experiment Station. Mention of a trade- ing mixture was adjusted to 1 x ligation buffer and 60- mark or proprietary product does not constitute a guarantee or 120 Weiss units of T4 ligase were added. The reaction was warranty of the product by USDA/ARS on Purdue University incubated at 37°C for 1 h and then 15°C for 12 h. The Abbreviations: bp, base pairs; kb, kilobase pairs; DNase I, deoxyri- DNA was gently extracted with phenol/chloroform and bonuclease I ; EDTA, ethylenediaminetetraacetie acid; RNAase TI, precipitated with isopropanol. The precipitate was resus- ribonuclease TI; SDS, sodium dodecyl sulfate; TLE, 10 mM Tris, pended in TLE and digested to completion with BamHI. 0.1 mM EDTA The digest was adjusted to 20 mM EDTA and loaded onto 108 a 5%-20% potassium acetate gradient that contained were sequentially subjected to RNAase T1 treatment (1 h 0.01% Sarkosyl. The sample was spun at 27,000 rpm for at 30 ° C), proteinase K digestion, phenol extraction and 15 h at 20 ° C in an SW28 Beckman rotor. Following centrif- ethanol precipitation as described by Melton et al. (1984). ugation the supernatant was aspirated and discarded. The The precipitate was dissolved in 50 lal of formamide loading pellet, consisting of annealed arms (30.2 kb), was dried and buffer, placed in a boiling water bath for 3 min, and 1 lal resuspended in 500 lal TLE. The DNA was extracted with samples were fractionated in a 6% polyacrylamide sequenc- phenol/chloroform, precipitated with isopropanol and re- ing gel. Bands were visualized by autoradiography carried suspended in 50 lal TLE. The purified, annealed arms were out overnight with intensifying screens. Size standards were aliquoted and stored at -20 ° C until used. used transcripts from HaeIII-digested pSP65, as well as SP6 Charon 35 arms (2.7 lag) and leaf DNA (0.8 lag) were control templates from New England Biolabs. ligated in a 20 lal reaction volume using 9 Weiss units of T4 DNA ligase. The ligation reaction was incubated at 12 ° C for 15 h and then 68 ° C for 5 min. The recombinant Results and discussion DNA molecules were either precipitated with ethanol be- The cultivar Raiden has been shown by electrophoretic and fore packaging or were packaged immediately into phage chromatographic analysis of its seed protein to lack the particles. Approximately one-half of a ligation reaction was A4, As and B~ polypeptides that are present in most other used for each packaging reaction. The library contained cultivars (Kitamura et al. 1984; Staswick and Nielsen 1983; approximately 5 x 105 independent plaque-forming units. Kitamura et al. 1980). Genetic studies have revealed that The Raiden genomic library was screened for Gy4 clones the absence of all three polypeptides results from a single, by the plaque hybridization procedure of Benton and Davis recessive allele designated gY4 (Kitamura et al. 1984; Scal- (1977) using as probe the nick-translated insert of a Gy4 lonet al. 1985). cDNA clone (pG258). Nitrocellulose filters were prehybri- Southern hybridizations of leaf DNA have shown that dized and hybridized at 42 ° C in 50% formamide, 5 x SSC, the Gy4 gene in the wild-type cultivar CX635-1-1-1 is on 5 x Denhardt's (0.1% bovine serum albumin, 0.1% Ficoll, a 13 kb EcoRI fragment and that the gene is present ap- 0.1% polyvinylpyrrolidone), 1% SDS, 50mM sodium proximately once per haploid genome (Scallon et al. 1985). phosphate, pH 6.5, 250 lag/ml denatured calf thymus DNA, Gys, a highly homologous glycinin gene which is also pres- 1 mM EDTA, 1 lag/ml poly(dG)-poly(dC), and 0.1 lag/m1 ent about once per haploid genome, exists on a 9 kb EcoRI linearized denatured pUC8 DNA. After hybridization the fragment in CX635-1-1-1. When a mixture of Gy4 and Gy5 filters were washed in 2 x SSC, 0.1% SDS at room tempera- sequences was used as a probe in Southern hybridizations ture and then in 0.1x SSC, 0.1% SDS at 66 °C for 5h. with Raiden genomic DNA, the same banding pattern was The filters were autoradiographed with an intersifier screen observed (e.g. 13 and 9 kb EcoRI fragments, data not for 1 to 2 days. shown). This indicated that the coding sequence for gY4 was present in the Raiden genome on the same size EcoRI DNA sequencing. Sequencing of DNA was performed as restriction fragment as in cultivars which produced the sub- described by Maxam and Gilbert (1980). DNA fragments unit. Hence, the failure of the null allele to produce the were end-labeled using either T4 polynucleotide kinase and subunit was not due to a complete deletion of the gene. ~-[32P]ATP (7,000 Ci/mmol) or the Klenow fragment and ~-[32p]dNTPs (3,000 Ci/mmol). Isolation of the Raiden Gy 4 gene RNA isolation. Poly(A) + RNA was isolated from poly- It was considered likely that the molecular lesion responsi- somes of mid-maturation stage embryos of Raiden and ble for the absence of the AsA~B3 subunit had occurred CX635-1-1-1 as described by Tumer et al. (1981). Total cel- in the structural gene. To investigate this possibility, a geno- lular RNA from mid-maturation stage embryos was iso- mic library of Raiden leaf DNA was constructed in the lated by the method of Hall et al. (1978). bacteriophage vector Charon 35 (Loenen and Blattner 1983). Approximately 2 × 105 independent plaque-forming Construction of pSP64/248HB and transcription of the anti- units from the library were screened prior to amplification sense Gy4 probe.