Lipochromosome mediated gene transfer: Identification and probable specificity of localization of human chromosomal material and stability of the transferents

Summary Using lipochromosomes (phospholipid-entrapped chromosomes) we have transferred the human HGPRT gene into HGPRT deficient mouse cells (A9) with a frequency of approximately 1×10⁻⁵ (Mukherjee et al., Proc. Natl. Acad. Sci. USA 75: 1361–1365; 1978). Two other genes located on the long arm of the human X-chromosome were also expressed in two independently derived populations of transferents (A9/GT3 and A9/GT4). We report here the chromosomal and enzymatic composition of human HGPRT-positive clones from each subpopulation analyzed in detail with alkaline Giemsa-11 staining. All the clones expressed human PGK and HGPRT, but one (A9/GT4C6) lacked human G6PD. In each of four clones examined microscopically, a small piece of presumptive human chromatin was visible in the karyotypes of most cells. The chromatin fragment was free or attached in each cell of an individual clone. When integrated, the human chromosomal fragment in each clone appeared associated with the centromere of the same telocentric A9 chromosome (No. 6 by Q-banding). These data suggest that: (a) substantial human chromosomal fragments can be transferred into recipient cells using the lipochromosome technique; (b) clones from human HGPRT positive A9 transferent subpopulations may or may not possess other human X-linked markers; (c) the stability of lipochromosomally transferred genes varied from clone to clone and stability is generally poor in the absence of continuous selection pressure (e.g., HAT); (d) when multiple X-linked human genes were transferred to mouse cells a cytologically detectable human chromosomal fragment was identified free or attached to a host chromosome; and (e) integration of transferred human chromosomal material into mouse chromosomes may occur at preferential site(s) in the recipient genome. This research was sponsored in part by the Office of Health and Environmental Research U.S. Department of Energy under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium

Summary We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in anin vitro preparation of bullfrog RPE-choroid. Changes in RPE [K⁺] _i alter the current produced by this pump. Increasing [K⁺] _o in the solution perfusing thebasal membrane increases RPE [K⁺] _i (measured with a K⁺-specific microelectrode), and also depolarizes theapical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0mm [K⁺] _o outside the apical membrane. Removal of Cl^– from the solution perfusing the basal membrane abolishes the K⁺-evoked apical depolarization by preventing the entry of K⁺ (as KCl) into the cell. We conclude that the increase in [K⁺] _i causes the decrease in pump current. This result is consistent with the finding that [K⁺] _i is a competitive inhibitor of the Na⁺–K⁺ pump in red blood cells.It is possible that the light-evoked changes in [K⁺] _o in the distal retina could alter RPE [K⁺] _i , and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.     

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.     

Linkage between low-density lipoprotein and igk light-chain genes in rabbits

The rabbit geneLpq, which codes for a low-density serum lipoprotein², is linked (34.6 ± 5.3 centimorgans) to the Ig kappa light-chain gene (Ab). There is no evidence thatLpq is linked to another gene,Prt, that was previously found to be linked to theAb gene. This suggests that the gene order for the three genes isPrt- Ab- Lpq. Abbreviations used in this paper Ig immunoglobulin - a the heavy-chain variable-region geneAa - b the kappa light-chain geneAb - q the low-density serum lipoprotein geneLpq     

Autoclavable Dispenser for Disposable Pipette Tips

An autoclavable dispenser for plastic disposable pipette tips is described that allows aseptic removal of individual tips while maintaining the sterility of the remaining tips.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Sperm-aggregating factor from Spisula oocytes

A membrane protein possessing sperm-aggregating activity was partially purified from Spisula oocyest. Spisula oocytes were incubated with three different media: A) 1 M urea, 5 mM EDTA, 10 mM Tris-HCI, pH 7.4, B) 1 M urea, 10 mM Tris-HCI, pH 7.4, and C) 5 mM EDTA in artificial sea water. Oocytes incubated in media A or B at 22°C were viable up to 15 min of treatment based on the trypan blue exclusion test. After this treatment period, oocyte viability gradually decreased as demonstrated by a progressive increase in the uptake of the dye. However, oocytes excluded the dye when incubated in medium C for 2 hr or longer. Oocytes incubated in medium A or B did not undergo germinal vesicle breakdown (GVBD) on exposure to sperm, while GVBD was induced on treatment with 70 mM KCI, suggesting removal or alteration of sperm receptors by the treatment. When sperm were incubated with oocyte extract prepared by treatment with medium A or B, they aggregated and formed clusters. The clusters remained unchanged for at least 1 hr at 22–24°C and sperm within the aggreates were motile. Extracts of Spisula oocytes showed species specificity by not agglutinating sperm of Arbacia punctulata, Asterias forbesi, ovalipes ocellatus, or Chaetopterus peramentaceus. The factor was puridied by ammonium sulfate fractionation (30% saturation) and by gel filtration on a Sephadex G 100 column. Four major protein peaks were eluted. Fraction comprising the second and third peaks possessed sperm-aggregating activity at an affective does od 2.5 μg of protein per ml. The factor is a heat-stable protein with an estimated molecular weight (mol wt) of 15 to 25 kdaltons.