Targeted disruption of the mouse Gz-alpha gene: a role for Gz in platelet function?

Gz is one of nine G proteins identified in platelets and its role in these cells is unknown. Our laboratory has generated a mouse deficient in the Gz-alpha gene in the hope of determining its in vivo function. Bleeding times from the tail tip of Gzalpha deficient mice was significantly longer than wild type mice. Platelet aggregation and ATP secretion did not differ between wild type and Gzalpha deficient mice. When mice were presented with a thromboembolism challenge no differences were observed in the survival or mortality of wild type or Gzalpha deficient mice, however a strain difference was observed. Ignoring the genetic background of a mutant mouse might lead to a misinterpretation of results and thus it is absolutely critical to take the genetic background into account when assessing any aspect of a mutant mouse.

A one-step quantitative reverse transcription polymerase chain reaction procedure.

Our laboratory has developed a one-step quantitative reverse transcription polymerase chain reaction (RT-PCR) procedure in which the reverse transcriptase enzyme and Taq DNA polymerase are combined in the one tube and a single, non-interrupted, thermal cycling program is performed. In the past, RT-PCR has been carried out with two separate steps: (1) reverse transcription of RNA to generate a cDNA pool and (2) polymerase chain reaction amplification of the cDNA. The two-step method can affect the accuracy of the procedure as the total number of manipulations is greater, thereby allowing a greater chance for pipetting errors. Quantitation by our method is achieved in a single reaction by the use of a competitive internal standard that is identical in sequence to the target RNA except for a deletion of 107 base pairs and uses identical primers and cycling conditions. Using this method, we have been able to quantify the amount of message of a G protein (G(zalpha)), in small amounts of tissue, such as dorsal root ganglia, from embryonic as well as postnatal mice.

Hypertolerance to morphine in G(z alpha)-deficient mice.

Our laboratory has generated a mouse deficient in the alpha (alpha) subunit of the G protein, G(z), (G(z alpha)) gene and we have examined the involvement of G(z alpha) in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50 degrees C or -5 degrees C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52 degrees C or 0.5 degrees C. Tolerance to morphine was induced in wild type and G(z alpha)-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and G(z alpha)-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the G(z alpha)-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and G(z alpha)-deficient brains. No changes in the affinity of morphine for its receptor or in the density of mu and delta opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of G(z alpha) does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of G(z alpha) delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.

Developmental expression of messenger RNA levels of the alpha subunit of the GTP-binding protein, Gz, in the mouse nervous system.

There has been recent evidence that Gz may play a role in the transmission of the neurotrophic signal from nerve terminals to the cell bodies [Johanson, S.O., Crouch, M.F., Hendry, I.A., Signal transduction from membrane to nucleus: the special case for neurons, Neurochem. Res. 21 (1996) 779-785]. We examined the developmental expression of the alpha subunit of Gz (Gzalpha) in the peripheral and central nervous systems of the mouse. Our laboratory has developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) for Gzalpha which makes use of a fragment of the PCR product shortened by 107 base pairs creating a standard which mimics the original RNA. Serial dilutions of the mouse RNA with a constant concentration of mimic RNA were made and the point where equal amounts of product are formed allows accurate measurement of Gzalpha mRNA in the tissue. We have demonstrated that in the developing mouse superior cervical ganglion (SCG), dorsal root ganglion (DRG) and trigeminal ganglion the expression of Gzalpha mRNA is highest perinatally. From 3 weeks of age, in all tissues with the exception of the SCG, Gzalpha mRNA levels fall to lower levels in the adult animal. The developmental pattern of expression of Gzalpha in both the cerebellum and the brain differs from the peripheral nervous system. In the cerebellum, Gzalpha mRNA expression is highest around birth and in the brain it is highest around third postnatal week and then the levels decline as adulthood is approached. These results suggest that the highest level of Gzalpha mRNA is expressed at the time when target tissue innervation is occurring. This further strengthens the hypothesis that Gzalpha is important in the transfer of information from target tissues to the innervating nerve cells.

Sequence similarity of phospholipase C with the non-catalytic region of src.

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. Here we report the cloning of a bovine brain complementary DNA encoding an enzyme PLC-148 that is characterized by calcium-dependent and phosphatidylinositol-specific phospholipase C activity when expressed in mammalian cells. Bovine brain messenger RNA contains a 7.5-kilobase transcript corresponding to the isolated cDNA; a related transcript of the same size is present in mRNA from some but not all human cell lines tested. Southern blot analysis of the bovine genome indicated that one or possibly two genes hybridize to the cloned PLC-148 cDNA. There is a striking sequence similarity between specific regions of PLC-148 and the non-catalytic domain of the non-receptor tyrosine kinases. The newly characterized crk transforming gene of the avian sarcoma virus CT10 also contains extensive sequence similarities with PLC-148.