Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

نویسندگان

  • K Malmström
  • M Krieger
چکیده

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature

We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-macroglobulin, compartmentalization of newly synthesized acid hydr...

متن کامل

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T....

متن کامل

Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild-type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a m...

متن کامل

Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification

After 4 h at 41 degrees C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell End1 and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. F., R. Fuchs, I. Mellman, and A. R. Robbins. 1986. J. Cell Biol. 103:2283-2297). We have measured recovery of function after return to the permissive temperature. Base...

متن کامل

Track detection on the cells exposed to high LET heavy-ions by CR-39 plastic and terminal deoxynucleotidyl transferase (TdT)

Background: The fatal effect of ionizing radiation on cells depends on Linear Energy Transfer (LET) level. The distribution of ionizing radiation is sparse and homogeneous for low LET radiations such as X or γ, but it is dense and concentrated for high LET radiation such as heavy-ions radiation. Material and Methods: Chinese hamster ovary cells (CHO-K1) were exposed to 4 Gy Fe-ion 2000 keV/...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • The Journal of biological chemistry

دوره 266 35  شماره 

صفحات  -

تاریخ انتشار 1991