Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides.

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.

Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS3B and rbcS3C promoter regions.

Sites of DNA-protein interaction were mapped in the promoter regions of two of the five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) in tomato. The two genes, designated rbcS3B and -3C, are actively transcribed in cotyledons of light-grown seedlings and in leaves, but are transcriptionally inactive cotyledons of dark-grown seedlings, in young and mature tomato fruit, and in roots. The combination and order of conserved DNA sequence elements in the promoter regions of the two genes are essentially identical, but differ considerably from that found in the promoters of the other three tomato rbcS genes, which show different transcription patterns. Nuclear extracts from cotyledons of 7-day-old tomato seedlings, and from leaves and young tomato fruit of mature plants defined multiple DNase I-protected sites in the promoter regions of both genes. The protection patterns were organ-specific, and encompassed previously identified conserved DNA sequence motifs as well as uncharacterized sequences. In contrast, nuclear extracts from mature tomato fruit and roots of 7-day-old seedlings failed to protect any of the promoter sequences, implying that DNA-binding proteins required for transcription of rbcS3B and -3C are inactive in these organs. These results are somewhat surprising since DNA-binding proteins from cotyledons of dark-grown seedlings and young fruit interact with the two promoters, although rbcS3B and -3C are not transcribed in these organs. The basis for transcriptional regulation of these two genes is discussed and the detailed pattern of DNase I protection in the promoter regions of the two genes is presented.

Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS1, rbcS2 and rbcS3A promoter regions.

DNase I footprinting assays were used to map sites of DNA-protein interaction in the promoter regions of three of the five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) in tomato. Organ-specific differences in DNase I protection patterns were observed using nuclear extracts derived from cotyledons, leaves, young fruit, mature fruit, and roots of tomato, implying that organ-specific transcription of these genes is controlled at the level of DNA-protein interaction. The three genes, designated rbcS1, -2 and -3A are similarly expressed in cotyledons of dark-grown seedlings, in immature tomato fruit, and in leaves under conditions of water stress. These three genes share at least three DNA sequence motifs, including the G-box sequence, which are apparently not present in the other two tomato rbcS genes. We find protection of one or more of these sequences in the aforementioned organs, indicating that the corresponding DNA-binding proteins could function in directing differential expression of the genes, although functional studies would be required to establish this point. While most of the DNase I protections encompass previously identified conserved sequence motifs and their flanking sequence, we also observe protection of additional sequences, many of which occur in the region of the transcription start site.

Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family.

The five genes encoding ribulose-1,5-bisphosphate carboxylase (rbcS) from tomato are differentially expressed. Transcription of the genes is organ specific and developmentally regulated in fruit and light regulated in cotyledons and leaves. DNase I footprinting assays were used to map multiple sites of DNA-protein interaction in the promoter regions of all five genes and to determine whether the differential transcriptional activity of each gene correlated with developmental or organ-specific changes in DNA-protein interactions. We show organ-specific differences in DNase I protection patterns, suggesting that differential transcription of rbcS genes is controlled at least in part at the level of DNA-protein interactions. In contrast, no changes were detected in the DNase I footprint pattern generated with nuclear extracts from dark-grown cotyledons versus cotyledons exposed to light, implying that light-dependent regulation of rbcS transcription is controlled by protein-protein interactions or modification of DNA binding proteins. During development of tomato fruit, most DNA-protein interactions in the rbcS promoter regions disappear, coincident with the transcriptional inactivation of the rbcS genes. In nuclear extracts from nonphotosynthetic roots and red fruit, the only detectable DNase I protection corresponds to a G-box binding activity. Detection of other DNA binding proteins in extracts from these organs and expression of nonphotosynthetic genes exclude the possibility that roots and red fruit are transcriptionally inactive. The absence of complex promoter protection patterns in these organs suggests either that cooperative interactions between different DNA binding proteins are necessary to form functional transcription complexes or that there is developmental and organ-specific regulation of several rbcS-specific transcription factors in these organs. The DNase I-protected DNA sequences defined in this study are discussed in the context of conserved DNA sequence motifs and previously characterized binding sites.

Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem.

The use of promoter-reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT-PCR analysis of RBS gene-specific transcript levels, it is shown that: 5' upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens.

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype.

Murine transfer factor. I. Description of the model and evidence for specificity.

A model for studying transfer of delayed-type sensitivity to mice with cellfree materials is described. The results with a particulate antigen (Candida) and 4 soluble protein antigens (PPD, ferritin, cytochrome c, and horseradish peroxidase) suggest that the phenomenon is antigen specific. Identical preparations from the spleens of insensitive donors were not active. This murine model should facilitate characterization of the immunologic and chemical properties of transfer factor.

Human tonsillar IgE biosynthesis in vitro. I. Enhancement of IgE and IgG synthesis in the presence of pokeweed mitogen by T-cell irradiation.

A study of the events regulating human IgE biosynthesis in vitro was undertaken with tonsillar lymphocytes. IgG synthesis was also studied to evaluate the specificity of our observations. T-cell irradiation significantly enhanced synthesis of IgE by pokeweed mitogen (PWM)-stimulated B cells from 12 of 18 donors and IgG in all 18 donors. This enhancement was the result of de novo immunoglobulin synthesis, since the amount of IgE and IgG spontaneously released from lysed and lysed-and-cultured mononuclear cells was significantly less than that detected in the cell cultures, and the augmentation was completely ablated by the treatment of the cells with cycloheximide or mitomycin C. Enhancement was also dependent on the presence of PWM; T-cell irradiation did not enhance IgE synthesis in unstimulated cultures. Moreover, this enhancement was also observed in the co-cultures of B cells and allogeneic irradiated T cells. These observations suggest that radiosensitive T cells exert a suppressive activity that contributes to regulation of human IgE and IgG synthesis and that the suppressor function as well as the helper function can overcome allogeneic disparities.

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato.

We have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2, are present as single genes at individual loci. Three genes, designated Rbcs-3A, Rbcs-3B and Rbcs-3C, are organized in a tandem array within 10 kb at a third independent locus. The Rbcs-2 gene contains three introns; all the other members of the tomato gene family contain two introns. The coding sequence of Rbcs-1 differs by 14.0% from that of Rbcs-2 and by 13.3% from that of Rbcs-3 genes. Rbcs-2 shows 10.4% divergence from Rbcs-3. The exon and intron sequences of Rbcs-3A are identical to those of Rbcs-3C, and differ by 1.9% from those of Rbcs-3B. Nucleotide sequence analysis suggests that the five rbcS genes encode four different precursors, and three different mature polypeptides. S1 nuclease mapping of the 5' end of rbcS mRNAs revealed that the mRNA leader sequences vary in length from 8 to 75 nucleotides. Northern analysis using gene-specific oligonucleotide probes from the 3' non-coding region of each gene reveals a four to five-fold difference among the five genes in maximal steady-state mRNA levels in leaves.

Characterization of the TrnD, TrnK, PsaA locus of Euglena gracilis chloroplast DNA.

The EcoRI fragment Eco J' of Euglena gracilis chloroplast DNA has previously been identified as a tRNA coding locus. The nucleotide sequence of a 2.3-kb region of the Eco J' fragment known to contain the tRNA genes has been determined. This locus was found to contain two tRNA genes, trnD-GTC and trnK-TTT. Separated from the trnK locus by a 43-bp spacer is an open reading frame of 398 codons. The open reading frame is 73-75% homologous to the amino-terminal coding regions of the spinach and maize genes for the P700 chlorophyll a apoprotein of photosystem I. It has been identified as exon I of an intron-containing psaA gene. The exon is followed by an intron of at least 214 bp that has features characteristic of other Euglena chloroplast introns. Major chloroplast RNA transcripts of sizes 5.5, 4.7, and 2.3 kb hybridize to a psaA-specific probe. The gene for a second photosystem I P700 apoprotein, psaB, has been located on an adjacent EcoRI fragment, Eco C.