Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.     

Multiple mRNAs encode peripherin, a neuronal intermediate filament protein.

Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp, specific for peripherin, a neuronal intermediate filament protein (IFP), have been isolated from a murine neuroblastoma cell lambda gt11 library by immunoscreening using peripherin antiserum. Antibodies eluted from the fusion proteins produced by clones 3u and 5g recognize the peripherin spots on immunoblots. Where they overlap the three cDNAs have identical sequences. cDNA 5g exhibits the closest homology to type III IFP cDNAs. cDNA 3u is identical to the corresponding region of cDNA 5g, except for the insertion of a 96 bp fragment at a position corresponding to the junction of exons 4 and 5 in type III IFP cDNAs. cDNA 5b is also identical to the corresponding region of cDNA 5g, except for the deletion of a 62 bp fragment at the junction of exons 8 and 9 in type III IFP cDNAs. S1 mapping experiments performed with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.     

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.     

Atrial natriuretic factor messenger ribonucleic acid and peptide in the human heart during ontogenic development

We have investigated the level of expression of the atrial natriuretic factor (ANF) gene in the human heart during ontogenic development by determining the concentrations of ANF messenger ribonucleic acid (ANF mRNA), of immunoreactive ANF (IR ANF) and of receptor reactive ANF (RR ANF), in myocardial samples of the various heart chambers. We found the level was high and almost identical in the left and right ventricles in utero. It gradually decreased during ontogenic development to reach the low adult levels, with a more rapid decrease in the right than in the left ventricle after birth. In the atria, ANF gene expression was high as early as the 13th week of gestation, was higher in the right than in the left atrium, and appeared little affected by ontogenic development.     

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.Abbreviations Atrazine 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine - diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NEU N-ethyl-N-nitrosourea - PSII photosystem II     

Mapping of Col3a1 and Col6a3 to proximal murine chromosome 1 identifies conserved linkage of structural protein genes between murine chromosome 1 and human chromosome 2q

We have investigated the degree of synteny between the long arm (q) of human chromosome 2 and the proximal portion of mouse chromosome 1. To define the limits of synteny, we have determined whether mouse homologs of seven human genes mapping to chromosome 2q cosegregated with anchor loci on mouse chromosome 1. The loci investigated were NEB/Neb, ELN/Eln, COL3A1/Col3a1, CRYG/Len-2, FN1/Fn-1, VIL/Vil, and COL6A3/Col6a3. Ren-1,2 and Acrg were included as two proximal mouse chromosome 1 anchor loci. The segregation of restriction fragment length polymorphisms at these loci was analyzed in the progeny of Mus spretus x C57BL/6J hybrids backcrossed to the C57BL/6J inbred strain. We found that five of the structural protein loci and the two anchor loci form a linkage group on proximal murine chromosome 1. The proposed gene order of this group of linked markers is centromere - Col3a1 - Len-2-Fn-1-Vil-Acrg-Col6a3-Ren1,2. Neb and Eln are linked neither to each other nor to any other marker on proximal mouse chromosome 1. Therefore, the mouse loci Col3a1 and Col6a3 are identified as flanking markers of the linkage group of structural protein loci. The estimated genetic map distances are Col3a1-13.3 cM-Len-2-3.4 cM-Fn-1-3.8 cM-Vil-9.6 cM-Acrg-2.1 cM-Col6a3-18.3 cM-Ren1,2. The available map information for human chromosome 2q markers and mouse chromosome 1 markers presented here tentatively identifies Col3a1 and Col6a3 as the border markers that define the limits of the syntenic chromosome segment. The order of mouse genes on chromosome 1 and their human homologs on chromosome 2q also appears to be conserved, suggesting that mapping of murine genes on the conserved segment may be useful to predict gene order in man.     

Chromium in plants

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1–4 μg/g compared to 0.5–1.5 μg/g).     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.