Molecular Basis for Allelic Polymorphism of the Maize GZobuZin - 1 Gene Faith C . Belanger ' and Alan

An abundant protein in maize (Zea mays L.) embryos is a storage globulin encoded by the polymorphic Glbl gene. Several Glbl protein size alleles and a null allele have been described. Here we report the isolation and nucleotide sequence analysis of genomic clones corresponding to two Glbl size alleles (Glbl-L and Glbl-S) and to the Glbl-0 null allele. The Glbl-L and Glbl-0 alleles differ from Glbl-S by the presence of small nucleotide insertions which are imperfect or perfect duplications, respectively, of adjacent sequences. In the case of Glbl-L, the insertion is in-frame and results in a protein larger than that encoded by Glbl-S, whereas in Glbl-0 the insertion causes a translational frameshift which introduces a premature termination codon. Although steady-state levels of Glbl-0 transcripts are extremely low in Glb l -0 /0 embryos, nuclear transcription assays indicate that the Glbl-0 gene is transcribed at a level comparable to that of Glbl-L. This suggests that the low amounts of Glbl-0 transcripts in the cytoplasm may be due to mRNA instability. G LOBULINS are the major storage proteins in maize embryos, accounting for 10-20% of the embryo protein (KRIZ 1989). The major globulin component, GLBl, is one of the most abundant proteins in normal mature embryos. GLBl is encoded by the single gene Globulin-1 ( G l b l ) for which several size alleles and a CRM(cross-reacting material) null have been described (SCHWARTZ 1979; OSTERMAN 1988). The three most common Glbl alleles have been designated L, I , and S for Large, Intermediate, and Small proteins, respectively. Several characteristics of Glbl make it an interesting system for study: (1) GLBl is an abundant protein encoded by a single gene (SCHWARTZ 1979), (2) expression of the Glbl gene is seed specific (BELANGER and KRIZ 1989) and (3) GLBl protein is not essential for seedling growth since homozygosity for the Glbl-0 null allele has no effect on embryo development, maturation, or subsequent germination (SCHWARTZ 1979). We previously reported the characterization of a cDNA clone for the Glbl-S allele (BELANGER and KRIZ 1989). Here we report the isolation and characterization of genomic clones for the Glbl-S, -L and -0 alleles.* Analysis of these clones has revealed the nature of allelic polymorphisms in Glb l . Nucleotide sequence comparisons indicate that he Glbl-L and Glbl-0 alleles are more closely related to each other than is either allele to Glbl-S, and it appears that the S allele is the progenitor from which the other two alleles are derived. Both the Glbl-L and 0 alleles differ University, New Brunswick, New Jersey 08903. reported here are X59084, X59083, and X59085, respectively. Genetics 129: 863-872 (November, 1991) ' Current address: Department of Crop Science, Cook College, Rutgers 'The EMBL accession numbers for the Glb l -S , -L, and -0 sequences from the S allele by small insertions within their respective protein coding sequences. In Glbl-L the insertion is in frame and results in a larger protein, whereas in the Glbl-0 allele the insertion causes a frameshift mutation in the amino-terminal region of the protein which is followed shortly by a premature termination codon. Premature termination of translation of Glbl-0 mRNA apparently results in transcript instability: from nuclear run-on experiments the level of transcription from Glbl-0 is similar to that of Glbl-L although the steady state level of the Glbl-0 transcript is barely detectable by northern blot analysis (BELANGER and KRIZ 1989). MATERIALS AND METHODS Materials: Embryos homozygous for the Glbl-L and Glb l S alleles were obtained from field-grown plants of the maize (Zea mays L.) inbred lines W64A and Va26, respectively, as previously described (KRIZ 1989). The Glbl-0 allele was originally identified in a Black Beauty popcorn line (SCHWARTZ 1979) and is maintained in a homozygous state by a combination of plant outcrossing and selfing. Embryos were harvested as previously described and frozen in liquid Nn. LambdaZAP vector arms, Gigapack packaging extracts, and exonuclease III/mung bean nuclease deletion kits were from Stratagene (La Jolla, California). w3'P-Labeled dATP (3000 Ci/mmol) was obtained from New England Nuclear (Boston, Massachusetts). NA45 paper was obtained from Schleicher and Schuell (Keene, New Hampshire). Gel-X tubes were obtained from Genex (Gaithersburg, Maryland). Protein extraction and immunoblot analysis: Preparation of protein extracts from mature maize embryos, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and immunoblot analysis were performed as previously described (BELANGER and KRIZ 1989; PUCKETT and KRIZ 199 1). Nucleic acid isolation: Preparation of total DNA from 864 F. C. Belanger and A. L. Kriz unfertilized ears was as described by DELLAPORTA, WOOD and HICKS ( 1 983) followed by further purification on CsCl gradients. Total R N A was isolated from frozen tissue by using the guanidine-HCl method described by COX ( 1 968). Polyadenylated R N A was fractionated from total R N A by oligo(dT)-cellulose chromatography (AVIV and LEDER, 1972). For use in hybridizations, cDNA fragments from appropriate clones were isolated by EcoRl digestion, separation on a l % agarose gel, and binding of the fragment to NA-45 paper. DNA fragments were labeled with [a-"'PI dATP by using a commercial random priming kit (BRL or Stratagene). Isolation and characterization of genomic clones: T o obtain a genomic clone corresponding to Glbl-L, a genomic library prepared from W64A nuclear DNA in the lambda vector Charon 32 (KRIZ, BOSTON and LARKINS 1987) was screened by using the Glbl-S cDNA clone as a radiolabeled probe essentially as described by HUYNH, YOUNG and DAVIS ( 1 985). Growth of recombinant phage in liquid culture and lambda DNA preparation were performed as previously described (KRIZ, BOSTON and LARKINS 1987). A single clone, designated XgClbl-L, of 17 kb was found to contain a 3.5kb BcoRI fragment which hybridized with the pcClblS probe. This fragment was cloned into the plasmid vector pBluescript SK and given the designation pgClbl-L. Genomic clones for Glbl-S and Glbl-0 were obtained by preparing size-selected libraries in the vector LambdaZAP. Southern blots of EcoRl digested DNA isolated from unfertilized ears of plants homozygous for each of the three alleles indicated that in all cases a 3.5-kb EcoRl fragment hybridized to the Glbl probe (see Figure IC). The D N A from this region of an agarose gel was purified using NA45 paper or Gel-X tubes as suggested by the manufacturers. This DNA was ligated to EcoRI-digested LambdaZAP arms and packaged by using the Gigapack system. The resulting libraries were screened by using the radiolabeled Glbl-S cDNA clone as probe. The genomic clones were excised from LambdaZAP as recombinant pBluescript SK(-) plasmids according to the manufacturer's protocols. For nucleotide sequence analysis, the genomic clones were subcloned into M 13mpl8 and mpl9 (YANISCH-PERRON, VIEIRA and MFSSINC 1985) to obtain inserts in opposite orientations. Overlapping unidirectional deletions corresponding to either strand were prepared from the appropriate M 13 clone RF by using a commercial exonuclease III/mung bean nuclease deletion kit (Stratagene). Dideoxynucleotide sequencing (SANCER, NICKLEN and COUISEN 1977) of single-stranded templates with T7 DNA polymerase was performed by using commercial sequencing kits (United States Biochemical Corp., Cleveland, Ohio; Pharmacia LKB Biotechnology Inc., Piscataway, New Jersey). The deoxyguanine triphosphate (dGTP) analog I-deaza dCTP was used to resolve GC compressions. Analysis of DNA sequences was performed on an IBM PC A T with either IBI Pustell Sequence Analysis software (International Biotechnologies Inc., New Haven, Connecticut) or DNAStar programs (DNAStar, Inc., Madison, Wisconsin). Mapping of 5' ends of transcripts: Primer extension analysis (KINGSTON 1989) was used to determine the 5' end of transcripts corresponding to each Glbl allele. A 27-base oligonucleotide homologous to the transcribed region from position 133 to 159 in the pcGlblS cDNA clone was endlabeled with [-y-"'P]ATP by using a commercial kit (BRL). The labeled primer was annealed to 1 pg of poly(A+) R N A isolated from 24 days after pollination (DAP) embryos homozygous for either the S or L alleles. To compensate for the low steady state level of Glbl transcripts in Glbl-O/ 0 embryos (BELANGER and KRIZ 1989), 16 pg of poly(A+) b " :--