Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.     

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

The antibody response against MART‐1 differs in patients with melanoma‐associated leucoderma and vitiligo

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma‐associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T‐cell immune responses directed against the melanocyte‐differentiation‐antigens MART‐1 (Melan‐A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART‐1 antibodies were only present in patients with MAL. Melanocyte antigen‐specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART‐1 differ between these diseases, which can help to differentiate MAL from vitiligo.     

The paramutagenic line niv-44 has a 5 kb insert, Tam 2, in the chalcone synthase gene of Antirrhinum majus

Summary Several alleles of the nivea locus of Antirrhinum majus, both stable and unstable, have been characterised genetically (Harrison and Carpenter 1973 a, b). In this work the niv-44 allele is characterised at the molecular level. It contains a 5kb insertion element, Tam 2, which has 14 base pair inverted repeats. There is a three base pair duplication at the target site, which is at the first intron-exon boundary of the chalcone synthase gene. Tam 2 homologous sequences are present in multiple copies in several A. majus lines, including niv-53, and most have at least a 2.9 kb sequence in common with the copy at the chalcone synthase gene. Possible reasons for the apparent stability of the niv-44 allele and molecular explanations for the role of this allele in paramutation in A. majus are discussed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Molecular analysis of paramutant plants of Antirrhinum majus and the involvement of transposable elements

Paramutation is observed when the Antirrhinum majus lines 44 and 53 are crossed. These two lines both have insertions at the nivea locus, which encodes chalcone synthase (chs). The allele niv-53 carries the transposable element Tam1 in the promoter region of the chs gene; niv-44 carries the element Tam2 within the gene. The Tam1 element has previously been extensively characterised. Here the Tam2 element is further characterised, and the arrangement of the nivea locus in paramutant plants is analysed. The complete sequence of Tam2, and that of a partial cDNA complementary to it, have been determined. The cDNA is probably transcribed from a different copy of Tam2 from that present at the nivea locus, and does not encode a functional protein. Genomic Southerns of F1 plants from the 53/44 cross show that no major rearrangements are consistently associated with paramutation at the nivea locus of A. majus. The isolation from a paramutant plant arising from a 53/44 cross of an allele (niv-4432) resulting from the excision of Tam2 is reported. The excision of Tam2 resulted in a 32 bp deletion of chs gene sequences. Plants homozygous for the new niv-4432 allele have white flowers and are still paramutagenic, demonstrating that Tam2 need not be present at the nivea locus for paramutation to occur. Different interactions between Tam1 and Tam2 are discussed, and a possible model for paramutation is presented.     

Isolation of homozygous transgenicBrassica napus lines carrying a seed-specific chimeric 2S albumin gene and determination of linkage relationships

UsingAgrobacterium tumefaciens-mediated gene transfer, 14 T0Brassica napus plants carrying one of three chimeric 2S albumin seed protein genes were obtained after co-transformation with theneo gene. Plants were made homozygous using either cell haploid culture or, for single-copy plants, traditional crossing methods. Sixty-five percent of kanamycin-resistant plants contained at least one copy of the seed protein gene, and multiple copies were usually at a single locus. In the majority of cases, at least one copy of theneo gene was linked to an introduced 2S albumin gene, demonstrating that co-transformation is not a reliable way to obtain lines without the marker gene linked to the gene of interest. In 3 T0 plants sequences derived from beyond the left border of the vector were integrated in the plant genome, in two cases partially rearranged, confirming that T-DNA insertion is not always precise. Procedures for efficiently determining this are described. This work highlights the extra steps needed to prepare transgenic lines for field studies and potential further breeding.     

Studies of the role of the propeptides of the Arabidopsis thaliana 2S albumin.

To investigate the possible roles of the Arabidopsis thaliana 2S albumin propeptides with respect to sorting, processing, and stability of the protein in plant cells, five gene constructions deleting or modifying the propeptides were made based on one of the genes encoding the Arabidopsis 2S albumin. These constructions were introduced into tobacco (Nicotiana tabacum) plants. Using subcellular fractionation and immunocytochemistry on ripe seeds, it was demonstrated that none of the propeptides was necessary for the sorting of the protein. Detailed protein-chemical analysis of the mature gene products indicated that, for all of the modified 2S albumin precursors made, the proteins were stably folded and correctly processed. However, the latter is less efficient when the internal fragment between the small and the large subunit is missing or when this internal fragment is changed. In an attempt to establish a rapid assay system for modified 2S albumin precursors, yeast cells were transformed with the same gene constructs. It was demonstrated that the processing machinery in yeast cells differs from that in plants, and, in a perhaps related observation, differences in stability of a particular modified protein were observed.