Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 alpha) results in partial combined 17 alpha-hydroxylase/17,20-lyase deficiency

Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.     

A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information.

A class of small RNA molecules possibly involved in RNA editing is present in the mitochondrion of Leishmania tarentolae. These "guide" RNA (gRNA) molecules are encoded in intergenic regions of the mitochondrial maxicircle DNA and contain sequences that represent precise complementary versions of the mature mRNAs within the edited regions. In addition, the 5' portions of several gRNAs can form hybrids with mRNAs just 3' of the preedited region. A model is presented in which a partial hybrid formed between the gRNA and preedited mRNA is substrate for multiple cycles of cleavage, addition or deletion of uridylates, and religation, eventually resulting in a complete hybrid between the gRNA and the mature edited mRNA.     

Guide RNA-directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.     

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.     

Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Genetic and Cytogenetic Analysis of the 43a-E Region Containing the Segment Polarity Gene Costa and the Cellular Polarity Genes Prickle and Spiny-Legs in Drosophila Melanogaster

A cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster is presented. Within this interval 27 complementation groups have been identified by extensive F(2) screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described.