Kathleen B. Bechtol

Derivation of mutant t-haplotypes of the mouse by presumed duplication or deletion.

The genetic properties of some new mutant t -haplotypes derived from the naturally occurring haplotype t 6 have been investigated. Several mutant haplotypes gave taillessness with brachyury, T , were viable when homozygous, and when in compound with t 6 , and all these expressed tf and appeared to have arisen by meiotic crossing-over between T and tf in the region of crossover-suppression of t 6 . The haplotype t h17 arose by crossing-over in a stock of t 6 carrying the translocation T(1; 17) 190Ca, in which the translocation break in chromosome 17 is within the crossover-suppressing region; hence t h17 could not be separated from T190. It was of the complementary crossover type in that it did not interact with T , and was lethal in compound with t 6 ; its male segregation ratio was normal with heterogeneity. A further haplotype, t h20 , expressed tf , gave taillessness with T , and was lethal when homozygous and in compound with t 6 . The expression of tf was attributed to a deletion, which also covered the loci of the nearby lethals knobbly, Kb , and t w5 . t h20 showed weak complementation of t w5 . The haplotype t h7 had previously been interpreted as carrying a duplication; it was shown that this duplication was semi-lethal when homozygous. it is suggested that lethal mutant t -haplotypes commonly arise by duplication or deletion but such structural changes are not necessarily at the end of the haplotype, and need not arise by unequal meiotic crossing-over.

Identification and redistribution of lamins during nuclear differentiation in mouse spermatogenesis.

Chromatin may be attached to the nuclear envelope through interaction of the nuclear membrane lamins A, B, and C. Such a hypothesis requires that these proteins are present in all cells with chromatin attachment to the nuclear envelope. We have investigated the distribution of the lamins during spermatogenesis in mouse, which exhibits extremes in nuclear envelope structural changes. By immunohistochemical techniques using human auto-antibodies and monoclonal antibodies against these molecules, we found that the lamins persist through all stages of spermatogenesis, though in highly variable amounts. They are also present during meiotic prophase (pachytene) when chromosomes are only locally attached to the nuclear envelope, analogous to the early prophase of somatic cells. Restructuring of the early spermatid nuclear envelope is accompanied by the appearance of a new lamin at the acrosomal fossa. In the epididymal spermatozoon the distribution of different lamins varies markedly over the nucleus suggesting special structural functions. The presence of lamins throughout spermatogenesis supports the concept that they are a general feature of the nuclear envelope structure, even where a lamina is not recognizable ultrastructurally.

Monoclonal Antibodies against Human Tumor-Associated Antigens

It is now well established that certain tumor cells express surface antigens that are not present on cells in the tissue from which the tumor was initially derived (Ruddon, 1978; Greaves and Janossy, 1978). Such antigens have several possible origins: (1) new genetic information resulting from either mutation or oncogenic viral infection, (2) expression of normal genetic information out of temporal or cellular context, such as the expression of fetal antigens on cells in an adult, (3) rearrangement or modification of normal cell surface structures by mechanisms such as glycosylation, protease activity, or intermolecular interactions due to changes in membrane structure or fluidity. The production of specific antibody reagents against these antigens would facilitate analysis of their structure and genetic origin and also provide a means of detection and perhaps even selective destruction of tumor cells (Table I).

H-2 typing of mutants of the t6 haplotype in the mouse.

Mutantt haplotypes derived from thet6 haplotype were typed forH-2. The mutantth2 that arose fromt6 due to crossing over in the region betweenT andtf had, as expected, lost theH-2 haplotype characteristic oft6. The haplotypesth17,th18, andtp1, which also arose by recombination, but which represent the complementary crossover products, including the distal part of thet6 haplotype, carried the sameH-2 type ast6. This suggests that crossing over betweentf andH-2 is suppressed inth17 andt18. This in turn suggests that mutantt haplotypes suppress crossing over for that part of thet chromatin that they still retain.The origin ofth7, which apparently did not include any crossover distal toT, and which retains the crossover-suppressing property oft6, retains thet6H-2 type. Unexpectedly, Jh20, which expressestf and was at first thought to have arisen due to crossing over, also retains theH-2 type oft6. This provides part of the evidence thatth20 arises fromt6 not by crossing over, but by a small deletion, and hence that duplication and deletion are possible modes of origin of mutantt haplotypes.

Germ-Cell-Related and Nervous-System-Related Differentiation and Tumor Antigens

In recent years increasing attention has been given to the cell surface as an important functional part of the cell. Its component molecular structure and the mechanisms for its involvement in the interactions of cells with their environment are currently the subject of intensive study. Considerable evidence suggests that important aspects of many complex cell-environment interactions involve specific components of the cell surface. A graphic example of this is the species-specific aggregation factors of sponge (Frazier and Glaser, 1979). The factor (molecule), eluted from the sponge cells in Ca2+, Mg2+-free seawater, will cause species-specific aggregation of dissociated sponge cells. While the role of this molecule in the intact sponge is unknown, it may function as one part of the complex system that maintains the cellular associations of the live sponge. Examples of specific surface function in higher organisms are tissue-specific aggregation of cells (Culp, 1978), the sorting out of cells from different tissues (e.g., mesonephric and chondrogenic cells) in cultures (Steinberg, 1978), and the requirement in kidney tubule induction for cell-surface contact between meta-nephrogenic mesenchyme and ureteric bud cells (Wartiovaara et al, 1974; Leh-tonen et al., 1975). Several mechanisms for cell-surface interactions have been proposed, but few of the interactions are well understood.

Monoclonal Antibodies Against Human Ejaculated Sperm-Application in Antibody Mediated Infertility

Antisperm antibodies occur commonly in the circulation of vasectomized males, in couples with idiopathic infertility and in animals hyper-immunized with human sperm. Many of these polyclonal antisperm antibodies are capable of inhibiting sperm function in vivo as well as in in vitro bioassays such as sperm fusion with the zona-free hamster egg. We are developing monoclonal antisperm antibodies (MASA) with the objective of defining a subset of surface antigens that are critical to sperm function and therefore could be involved in human infertility. Sperm membranes, employed as immunogens, were isolated from washed ejaculated sperm following nitrogen cavitation and repeated centrifuga-tion. Spleen cells from immunized BALB/c mice were fused with HAT-sensitive myeloma cells (SP2/0-Agl4) and antisperm antibody activity was detected in the resultant hybridoma supernatants by a solid phase RIA using washed, intact sperm as the antigen source. Thirteen positive clones have been isolated, 6 producing immunoglobulins of the mu class and 7 of the gamma class. Characterization of several of these MASAs as to antibody and antigen concentration dependency in the RIA indicates that their behavior is highly individualistic. These MASAs are species but not tissue specific. MASA binding to human sperm has been localized to distinct surface domains by indirect immunofluoresence.