Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5.

Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II. This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27). More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III. This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively.

Two small subunits in Arabidopsis RNA polymerase II are related to yeast RPB4 and RPB7 and interact with one another.

An Arabidopsis cDNA (AtRPB15.9) that encoded a protein related to the RPB4 subunit in yeast RNA polymerase II was cloned. The predicted molecular mass of 15.9 kDa for the AtRPB15.9 protein was significantly smaller than 25 kDa for yeast RBP4. In SDS-PAGE, AtRPB15.9 migrated as the seventh or eighth largest subunit (i.e. apparent molecular mass of 14-15 kDa) in Arabidopsis RNA polymerase II, whereas RPB4 migrates as the fourth largest subunit (i.e. apparent molecular mass of 32 kDa) in yeast RNA polymerase II. Unlike yeast RPB4 and RPB7, which dissociate from RNA polymerase II under mildly denaturing conditions, plant subunits related to RPB4 and RPB7 are more stably associated with the enzyme. Recombinant AtRPB15.9 formed stable complexes with AtRPB19.5 (i.e. a subunit related to yeast RPB7) in vitro as did recombinant yeast RPB4 and RPB7 subunits. Stable heterodimers were also formed between AtRPB15. 9 and yeast RPB7 and between yeast RPB4 and AtRPB19.5.

Reconstitution of yeast and Arabidopsis RNA polymerase alpha-like subunit heterodimers.

Two subunits of about 36-44 kDa and 13-19 kDa in the eukaryotic nuclear RNA polymerases share limited amino acid sequence similarity to the alpha subunit in Escherichia coli RNA polymerase. The alpha subunit in the prokaryotic enzyme has a stoichiometry of 2, but the stoichiometry of the alpha-like subunits in the eukaryotic enzymes is not entirely clear. To gain insight into the subunit stoichiometry and assembly pathway for eukaryotic RNA polymerases, in vitro reconstitution experiments have been carried out with recombinant alpha-like subunits from yeast and plant RNA polymerase II. The large and small alpha-like subunits from each species formed stable heterodimers in vitro, but neither the large or small alpha-like subunits formed stable homodimers. Furthermore, mixed heterodimers were formed between corresponding subunits of yeast and plants, but were not formed between corresponding subunits in different RNA polymerases from the same species. Our results suggest that RNA polymerase II alpha-like heterodimers may be the equivalent of alpha homodimers found in E. coli RNA polymerase.

A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two alpha-motifs flanked by a highly charged C terminus.

We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13-15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the alpha subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.

Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II.

Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II.

Arabidopsis expresses two genes that encode polypeptides similar to the yeast RNA polymerase I and III AC40 subunit.

A 40-kDa subunit in eukaryotic RNA polymerases (Pol) I and III (e.g., yeast yAC40) is related in a part of its aa sequence to the alpha subunit of prokaryotic Pol and to a 35-44-kDa subunit in Pol II (e.g., yeast yB44). We have cloned two cDNAs, AtRPAC42 and AtRPAC43, from an Arabidopsis thaliana (At) (ecotype Columbia) lambda Yes expression library that encode Pol I and III subunits related to yAC40. The aa sequences derived from the cDNA clones were found to be 72% identical to each other and 40% identical to yeast Pol I and III subunits yAC40, but only 30% identical to yeast Pol II subunit yB44. While most other nuclear Pol genes identified to date are single-copy genes, two genes encode 42 and 43-kDa subunits of At Pol I and/or III. A 42-kDa subunit with identical mobility in SDS-PAGE to the aAC42 in vitro translated subunit is found in Pol III purified from At suspension culture cells.

Obstetric forceps pad designed to reduce infant trauma.

The use of the standard obstetric forceps has been associated with varying degrees of maternal and fetal trauma. To reduce the degree of skin markings, a pliable polyurethane pad with selfadherent backing has been designed, which can be applied to each blade of the forceps. One hundred five neonates who required forceps-assisted deliveries were observed for evidence of skin trauma immediately after delivery and again at 24 hours. Padded forceps significantly reduced craniofacial visible skin markings when compared with the skin markings produced by the unpadded forceps. The addition of the pad to the forceps blade had no adverse effect on the mother, and obstetricians encountered no problems in the application and use of the pads.

Changes in the management of severely Rh-immunized patients.

Since July 1, 1978, we have instituted the following changes in the management of severely Rh-immunized patients: (1) serial amniotic fluid optical density (delta OD450 ) values and real-time ultrasound scanning beginning at 21 weeks' gestation, (2) fetal transfusions as early as the twenty-third week, (3) ultrasound surveillance during and after intrauterine transfusions, and (4) planned premature delivery with neonatal exchange transfusions for selected cases between 29 to 32 weeks and for all patients after 32 weeks' gestation. The perinatal survival rate (83.8%) in 37 isoimmunized pregnancies managed with this regimen was significantly higher (p less than 0.05) than in 34 such pregnancies (55.9%) managed according to the protocol utilized during the previous four years.

Ultrasound air contrast transfusion technique: an aid to managing the Rh-affected fetus with ascites.

The successful management of an Rh-sensitized pregnant patient is reported in whom fetal intrauterine transfusions were utilized despite the presence of fetal ascites before the 26th week of gestation. A new ultrasound air contrast technique is described that confirms the correct intraabdominal placement of the transfused blood. The technique is not dependent on the amount of blood introduced, nor is it masked by fetal ascites.

Dysmenorrhea: treatment with an antiprostaglandin.

Painful menses, one of the most frequent gynecologic complaints, is incapacitating for many women. It has recently been proposed that increased endometrial prostaglandin production and prostaglandin-induced myometrial contractility may be responsible for dysmenorrhea. In this prospective, double-blind, 3-way, crossover study, relief of pain by an antiprostaglanding drug, ibuprofen (400 mg), was compared with propoxyphene (64 mg) and placebo in 22 women with severe primary dysmenorrhea. Ibuprofen was significantly more effective in 18 patients when compared to the other 2 treatment regimens (P less than 0.001), while propoxyphene was superior to placebo in 13 patients (P less than 0.05). Prostaglandin E and F synthesis rates in endometrial biopsy specimens taken on the second day of treatment in 2 patients during each treatment cycle were lowest during ibuprofen in one case but showed no definite pattern in the second.

Nonlinear kernels of the human ERG.

In this paper we examine the use of a symmetric binary random stimulus for eliciting the ERG, and for calculating the first-order and second-order kernels of a nonlinear functional expansion of the response. We show that if the stimulus is represented in a non-dimensional form, then the units in which all kernels are measured are the same as the units used to measure the response, microvolts in the case of the ERG; further, contributions from all kernels to the response can be added without scale factors. We present the first-order and second-order kernels measured for a population of 15 normal subjects in a clinical setting. The measurements were made at various levels of adaptation ranging from photopic to scotopic conditions. The second-order kernels illustrate the processes of rapid adaptation (less than 100 ms) in the retina.