S. Packman

Globin gene expression in cultured erythroleukemic cells

Abstract A cultured, murine erythroleukemic cell line, which initially contains no detectable hemoglobin, can be induced to synthesize hemoglobin in quantities comparable to those found in normal red blood cells. In order to distinguish between several molecular mechanisms which might explain this induction, radioactive DNA complementary to mouse globin messenger RNA was used as a hybridization probe to measure globin genes and mRNA quantitatively during this form of differentiation. The results indicate that the number of globin genes does not change as these cells accumulate hemoglobin and that there are less than five copies of the globin genes per haploid genome. On the other hand, differentiated cells accumulate, on the average, 7000 to 8000 molecules of globin mRNA per cell, compared with less than ten in each undifferentiated cell. The accumulation of globin mRNA ceases in the presence of actinomycin D, suggesting that it is dependent on de novo RNA synthesis. It is also inhibited by cycloheximide and puromycin, suggesting a requirement for continued protein synthesis. Although a mechanism involving the post-transcriptional stabilization of newly synthesized globin mRNA can not be ruled out, these results are most simply explained on the basis of transcriptional activation of globin genes. Further data suggest that the globin mRNA synthesized in these erythroleukemic cells is relatively stable (chemical half-life more than 10 h) and is indistinguishable from authentic reticulocyte globin mRNA. The amount of globin mRNA, synthesized at the rate of approximately 20 nucleotides per second, has been measured and found comparable to the amount in mouse reticulocytes.

A comparison of globin genes in duck reticulocytes and liver cells

Abstract Synthetic duck globin DNA has been used to compare the number of genes corresponding to globin in duck reticulocytes and liver cells. The results indicate that the number of globin genes does not differ significantly in these organs and suggest, therefore, that neither amplification nor extensive reiteration of these genes occurs as these two cell types become committed to divergent lines of development.

THE ORGANIZATION AND DIVERSITY OF IMMUNOGLOBULIN κ AND λ GENES

Abstract: Our studies have focused on several hypotheses put forward to account for the diversity of immunoglobulin genes. These studies have shown that the constant regions of the mouse κ and λ chains are represented as relatively unique sequences in the genome of the mouse. Experiments which have attempted to focus on the genetic representation of the variable region of the κ chain are not as readily interpreted. They do, however, rule out thousands of closely related germ line sequences. As a result of ambiguities in the κ system, we have turned our attention to a simpler model, the mouse λ light chain. Since detailed analyses have shown that there are at least seven very closely related λ light chains, this class provides a very useful model for studies involving molecular hybridization. Utilizing mouse λ chain 3 H-cDNA, hybridization kinetic analyses indicate that these sequences are represented among the relatively unique genetic sequences in the mouse genome. This result tends to rule out a germ line model for antibody diversity. The reservations with respect to reaching such a conclusion are also discussed.

THE ORGANIZATION OF IMMUNOGLOBULIN GENES

ABSTRACT In order to distinguish among various models which have been advanced to account for the diversity of antibody molecules, we must know how the constant and variable regions of immunoglobulins are represented in the somatic genome. For this purpose, we have purified mRNA corresponding to a mouse kappa immunoglobulin light chain from the myeloma tumor, MOPC-41. This mRNA directs the enzymatic synthesis of highly radioactive DNA (cDNA). This cDNA, which should correspond to the constant region of the kappa chain, was assessed for reiteration frequency using hybridization kinetic analysis and was found to be represented approximately three times per haploid genome . This result tends to rule out germ line hypotheses which require many copies of the constant region gene. It also requires the postulation of a recombinational mechanism to join constant and variable region sequences. Hybridization kinetic analyses designed to assess the entire light chain sequence (C and V regions) made use of 125I-MOPC-41 mRNA. These revealed a major component of relatively unique frequency and a minor (~ 20%) component with a reiteration frequency of approximately 30-50 copies per haploid genome. Careful analysis of the extent of hybridization of this mRNA to DNA prepared from several tumors and tissues, thermal profiles, and relevant competition studies, while sensitive, do not permit us to distinguish unambiguously between a germ line model and the type of somatic mutation model which permits germ line genes corresponding to each kappa subgroup. Our results do, however, clearly rule out the existence of thousands of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive, stable cross hybridization.

Induction of Globin mRNA in Friend Leukemia Virus-infected Cells and Its Presence in Viral 60S RNA

The Friend leukemia virus (FLV)-infected cell line, T-3-Cl-2, undergoes a form of erythroid differentiation in culture when treated with an appropriate inducer, such as dimethylsulfoxide ((CH3)2SO). Thus, whereas untreated cells contain no detectable hemoglobin, treated cells accumulate hemoglobin in quantities comparable to those in the mature mouse red blood cell. We have investigated the mechanism of hemoglobin induction by quantitating the number of globin genes and the amount of globin mRNA in cells before and during the period of hemoglobin accumulation. The results indicate the number of globin genes does not change as the cells accumulate hemogtobin: There are less than 5 globin genes per haploid genome. On the other hand, whereas cells lacking hemoglobin contain little, if any, globin mRNA, hemoglobin-containing cells accumulate, on the average, 8,000 molecules of globin mRNA per cell. The most direct, although, by no means, the only interpretation of these results is that the induction of hemoglobin synthesis involves transcriptional activation of the globin genes. Using this same cell line, we show that mouse globin mRNA sequences are also present in viral particles purified from the culture medium of globin-producing cells. These globin mRNA sequences are absent from viral particles derived from T-3-Cl-2 cells which are not producing globin mRNA. Virus-associated globin mRNA sequences sediment in association with 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60 S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin mRNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin mRNA does not become associated with the 60S viral RNA complex; and that globin mRNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin mRNA. The presence of globin mRNA sequences in FLV particles has important implications in terms of our ability to distinguish between host and viral RNAs in viral particles and in terms of the possible role RNA tumor viruses might play in transduction of genetic information.

DNA Complementary to Globin and Immunoglobulin mRNA: A Probe to Study Gene Expression

During the development of an embryo, various processes occur which induce the formation of differentiated tissues. Some of these processes can be observed at a molecular level, for example, myosin is synthesized in muscle cells, crystaline in lens tissue, keratin in skin, immunoglobulin in lymphocytes and hemoglobin in reticulocytes (1,2).