Orcein staining and the identification of polytene chromosomes.

Acetic orcein staining of polytene chromosomes was introduced in 1941 shortly after the initial studies on aceto-carmine-stained chromosomes by Bridges (2) and has remained a standard method of preparation. Orcein dye can be purchased in both its natural form as extracted from two species of lichens, Rocella tinctoria and Lecanora parella, and a synthetic form. The mechanism of staining is not clearly understood because the stain itself is a variety of phenazones, which may interact at an acid pH with negatively charged groups or possibly interact hydrophobically with chromatin. Acetic acid fixation accommodates stretching of the chromosomes in the interband regions during a squash, thus providing for a higher resolution of the banding structure. The later addition of lactic acid to aceto-orcein (3) kept the glands softer in the fix and allowed for easier spreading of chromosomes. The method and its variations have appeared more recently in several publications (4,5). Drosophila polytene chromosomes are found in a number of larval tissues, including the midgut, hindgut, and the fat body, but the largest chromosomes are found in the salivary glands of the third instar. They are referred to as interphase chromosomes and are structurally more comparable to highly amplified interphase chromatin than to mitotic chromosomes because the gland grows by endoreplication of DNA, thus increasing cell size rather than cell number. Each of the homologs is tightly synapsed in this somatic tissue and undergoes approx 10 rounds of endoreplication, producing 1024 chromatids closely associated in parallel arrays.

Transformation of MC3T3-E1 cells following stress and transfection with pSV2neo plasmid.

A continuous cell line, MC3T3-E1 cells, originally derived from murine calvaria bones, loses its osteogenic properties as a result of extended passage number under stress conditions. These aged/stressed MC3T3-S cells, although nontumorigenic, do not display some of the osteogenic properties characteristic of the MC3T3-E1 cells. Altered properties include low expression of alkaline phosphatase, diminished collagen synthesis and inability to form mineralized nodules in vitro. We attempted to reactivate these osteogenic properties by transfections with a pSV2neo plasmid containing the TGFbeta1 gene. During these experiments we found that transfected MC3T3-S cells not only acquired high alkaline phosphatase activity and a potent mineralization potential, but also properties akin to the transformed state, such as ability to grow in soft agar and ability to produce tumors in immunodeficient animals. Further analysis showed that the TGFbeta1 gene is not required and that the changes can be introduced by transfections with pSV2neo alone. In contrast, MC3T3-S cells transfected with pcDNA3 (a plasmid containing only the SV40 origin of replication, early promoter, enhancer and polyadenylation signals) or mock-transfected MC3T3-S cells did not show any transformation traits. The results identify two additional SV40 fragments present in pSV2neo (SV40 virus sequence; Genbank accession number: NC_001669: 4100-4191 and 2668-2774) as functional elements contributing to the transformation of aged/stressed and immortalized osteoblastic cells. These findings are analogous to earlier reports describing the cell modifying potential of pSV2neo. We conclude that stressed and aged MC3T3-S can be transformed by transfection with pSV2neo and that such cells acquire not only the tumorigenic potential but exhibit also some of the osteogenic properties characteristic of the parent MC3T3-E1 cells.