Acid precipitation of mammalian cell fermentation broth.

The broth harvested from the "fermentation" of mammalian cells contains many contaminants produced by the cells, including nucleic acids, proteins, and complex polysaccharides, in addition to the product. These contaminants can foul filtration membranes, precipitate during storage or processing steps, and interfere with the performance of chromatographic separations. Acidification of hybridoma cell fermentation broth at the time of harvest from the fermenter has been investigated as a method of selectively precipitating the major contaminants from the soluble antibody product. Between pH values of 6.0 and 4.5, precipitation of the major contaminants is rapid and independent of the temperature (4-37 degrees C), with less than 10% of the antibody coprecipitating in 4 of 5 cases. Antibody activity and physical characteristics were found to be unaltered above a pH of 3.8. Recovery of antibody at the stage of concentrated (50 x), diafiltered bulk was improved from 63% to 84% by using the acid precipitation step. An additional benefit is that retrovirus is effectively inactivated by incubation at a pH below 4.2.

Species specificity of iron delivery in hybridomas.

Studies with Human X Human (H X H), Human X Mouse (H X M), and Mouse X Mouse (M X M) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that H X H hybridomas do not respond to bovine transferrin a+ concentrations up to 100 micrograms/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. H X M and M X M hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on H X H hybridomas but was ineffective on H X M hybridomas. This demonstrated the functionality of the human transferrin receptor in H X H hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in H X M hybridomas. H X H and H X M hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. M X M hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.

Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell.

We describe the first example of a predominantly nuclear protein which during mitosis becomes part of the mitotic apparatus. This protein has been termed the nuclear-mitotic apparatus (NuMA) protein. In interphase cells NuMA protein is restricted to the nucleus and is a constituent of isolated nuclear matrices, but in mitotic cells it is observed by indirect immunofluorescence microscopy to be concentrated at the polar regions of the mitotic apparatus. This mitotic localization is dependent on the integrity of the spindle, since treatments which disrupt the spindle result in dispersion of NuMA protein throughout the cell. Comparison to the subcellar distribution of tubulin at different stages of the cell cycle indicates that NuMA protein is distinct from the previously identified components of the mitotic spindle. Its association with the nuclear matrix and its localization during mitosis to the site of nuclear reassembly suggest the interesting possibility that NuMA protein could be representative of a class of proteins involved in the early events of nuclear reassembly. NuMA is present in the nuclei and mitotic spindle of all types of human cells that have been examined, but proteins of similar molecular weight (300,000 daltons in dissociating solvents) or immunological specificity are not detected in cells of other species (including monkey). However, the NuMA protein is synthesized in a human/Chinese hamster hybrid cell containing a reduced number of human chromosomes. Immunofluorescence studies of this hybrid cell showed that the distribution of NuMA protein is equivalent to that in human cells. These results suggest that the human gene coding for NuMA protein, unlike other genes coding for human specific nuclear proteins, can be expressed in human/hamster hybrid cells and that the cell hybrids will be useful in further characterization of NuMA protein.

Expression of genes coding for non-histone chromosomal proteins in human-Chinese hamster cell hybrids. An electrophoretic analysis.

The non-histone chromosomal (NHC) proteins of several human and hamster cell lines as well as human-hamster cell hybrids were investigated by one- and two-dimensional gel electrophoresis. In each type of human cell, over 90% of the 230 most prominent NHC proteins were indistinguishable electrophoretically from the NHC proteins of the hamster cells. Of the roughly 10% which were distinct, 11 of the proteins may be human-specific, since they were found in each type of human cell examined. The NHC proteins in seven different stable human-hamster cell lines, which, in total, contained markers for 21 of the 24 human chromosomes, were electrophoretically indistinguishable from those of the parental hamster cell line, with the exception of a single protein in one hybrid line. It is concluded that many of the genes coding for the apparently human-specific NHC proteins are not expressed in these hybrid cells. The single exception to this appeared to be a gene coding for a protein of roughly 300,000 daltons. This protein was synthesized in one of the hybrids, as well as in each type of human cell. Since the NHC proteins of the hybrid and hamster cells were virtually identical, there was no evidence for the expression of genes coding for NHC proteins characteristic of the differentiated state of the parental human cells.

Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation.

The structure of the bacterial chromosome was investigated after introducing breaks in the DNA with gamma irradiation. It is demonstrated that irradiation of the chromosome in the cell prior to isolation results in partial unfolding of the isolated condensed DNA, while irradiation of the chromosome after it is released from the cell has no demonstrable effect on DNA folding. The results indicate that RNA/DNA interactions which stabilize DNA folds are unstable when breaks are introduced in the DNA prior to isolation of the chromosome. It is suggested that the supercoiled state of the DNA is required for the initial stabilization of some of the critical RNA/DNA interaction in the isolated nucleoid. However, some of these interactions are not affected by irradiation of the cells. Remnant supercoiling in partially relaxed chromosomes containing a limited number of DNA breaks has the same superhelical density as the unirradiated chromosome. This suggests that restraints on rotation of the packaged DNA are formed prior to the physical unwinding which occurs at the sites of the radiation induced DNA breads. - Analysis of the in vitro irradiated chromosomes shows that there are 100 +/- 30 domains of supercoiling per genome equivalent of DNA. The introduction of up to 50 double-strand breaks per nucleoid does not influence rotor speed effects of the sedimentation coefficient of the chromosome.

Ultraviolet induction of prophage lambda during inhibition of deoxyribonucleic acid synthesis by hydroxyurea.

Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) precursors and causes the cessation of DNA synthesis. It did not cause induction of lambda. Superinfection of an irradiated lysogen with lambdaind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of lambda prophage to occur.