SummaryWe have resolved B220+IgM -B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen) . Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system . Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7 -pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFLlb.l, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D -J rearrangement, but no V-D-J . Finally, functional analysis demonstrates that the proliferative response to ID7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A .

The complete nucleotide sequence of the transferred region (T-DNA) of an octopine tumor inducing (Ti) plasmid fromAgrobacterium tumefaciens (pTi15955) has been determined. A total of 24 595 nucleotides extending approximately 900 bases to either side of the outermost, T-DNA boundaries was sequenced. Computer analysis of the sequenced portion of the Ti plasmid revealed that recognition sites for 72 restriction endonucleases are present in the DNA sequence at least once; no site forEcoK exists in this DNA sequence. Two imperfect 24 base repeats border the T-DNA sequence; the left starts at position 909 and the right ends at position 23 782, giving the T-DNA region a total length, of 22 874 nucleotides. Another two similar 24 base repeats lie within T-DNA and divide it, into three distinct domains: T-left (TL-DNA) 13 175 bp of apparently eukaryotic origin; T-center (TC-DNA) 1816 bp of prokaryotic origin; and T-right (TR-DNA) 7 883 bp of eukaryotic origin. The T-DNA contains nine reported transcripts, however, 26 open reading frames longer than 300 bases that start with an ATG initiation codon were found. Fourteen open reading frames are bounded by putative eukaryotic promoters, ribosome binding sites, and poly(A) addition sites and occur only in TL-and TR-DNAs. No open reading frames showing eukaryotic promoter sequences are located within the TC-DNA.

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.

Recombinant phage X177.4 contains a gene for j3 phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-Bam}Il fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.The transfer of foreign genetic information to broad-leafed plants by means (4) and maintained on Murashige and Skoog medium (MS) without hormone supplement. The tissue was cloned by the feeder plate method (9) and then placed in liquid culture to induce shoot-stem elongation (10). The shoots were grafted (11) onto 6-to 8-week-old N. tabacum var. Xanthi plants and grown at 22°C with a 16-hr photoperiod. Flowers were self-pollinated, and the seeds were allowed to mature.Quantitative and Qualitative Protein Assays. Proteins were extracted and quantified as described (8). Phaseolin was quantitated in tissues by dot-immunobinding assay (12). Protein patterns were analyzed after fractionation on a 13% polyacrylamide gel (13) or by two-dimensional gel electrophoresis (14) followed by electrophoretic immunoblot analysis with polyclonal antiserum to phaseolin (15). Antigenantibody complexes were visualized by treating the filters with 251I-labeled Staphylococcus protein A, followed by autoradiography.For immunodetection of concanavalin A-bound proteins, tissue extracts were incubated with concanavalin ASepharose beads followed by elution of the bound fraction with 1% NaDodSO4. The bound and unbound fractions were subjected to NaDodSO4/polyacrylamide gel electrophoresis followed by immunoblot analysis as described earlier.Isolation of RNA and Blot-Hybridization Analysis. Total RNA was isolated from leaves as described (16). RNA from developing seeds was prepared by isolation of polysomes followed by phenol extraction of the polysome pellet (17 3320The publication costs of this article were defrayed in part by page charge pay...

Zeins, the seed storage proteins of maize, are a group of alcoholsoluble polypeptides of different molecular masses that share a similar amino acid composition but vary i n their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal i s to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whelher this protein would be stable i n nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, p-phaseolin, in nonseed tissues and to determine the basis for i t s stability/instability. We have introduced the 15-kD zein and bean p-phaseolin-coding sequences behind the 35s cauliflower mosaic virus promoter into tobacco (Nicofiana fabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein i s stable not only i n seeds but in all nonseed tissues tested, whereas the P-phaseolin protein accumulated only i n mid-and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the p-phaseolin protein i n nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention i n the ER or to the proteaseresistant nature of the protein.Seed storage proteins constitute a potentially useful class of proteins for the improvement of forage crops if they can be made to accumulate in leaves. It is a fairly simple genetic engineering feat to introduce a seed protein-coding sequence behind a strong constitutive promoter into transgenic plants and ensure high rates of synthesis of the corresponding protein. However, stability of the protein in an alien environment is still not clearly defined and has to be treated on a case by case basis.Seed proteins are synthesized during seed development and accumulate in protein bodies. These proteins are then utilized by the emerging seedling during germination. The major seed protein in dicotyledonous plants are the saltsoluble globulins, which are stored in vacuole-derived protein bodies. Most monocot seed storage proteins are '