R. M. Zsigray

TheB locus (MHC) in the chicken: Association with the fate of RSV-induced tumors

The fate of tumors induced by Rous sarcoma virus (RSV) was determined in anF2 population segregating at three alloantigen loci. TheF1 resulted from crossing tumor-resistant RPRL line 61 (B2B2D3D3I2I2) with tumor-susceptible RPRL line 151 (B5B5D4D4I8I8). Among theF2 segregantsB2B2,B2B5, andB5B5, the percentage of chicks dying of terminal tumors (by 70 days post-inoculation) was 5, 26, and 93, respectively (P≦0.01). NeitherD orI genotypes nor sex significantly affected tumor growth. In chickens with terminal tumors, the incidence of metastatic lesions was also significantly associated withB genotypes. Thus, the MHC chromosomal region in the chicken appears to exert a crucial role in determining the outcome of RSV-induced tumors.

Electro-transformation of Clostridium beijerinckii NRRL B-592 with shuttle plasmid pHR106 and recombinant derivatives

Conditions for transformation of the solventogenic anaerobe Clostridium beijerinckii NRRL B-592 with plasmid DNA via electroporation are described. Shuttle plasmid pHR106 and two derivatives constructed in this study were transferred and were expressed in this organism. One recombinant derivative of pHR106 was constructed by separately subcloning the clostridial tetracycline (tetP) resistance genes into pHR106. The second vector conferring erythromycin resistance was obtained via in-vivo recombination. The new constructs, termed pRZL and pRZE respectively, were then transferred to C. beijerinckii in order to evaluate their potential as shuttle vectors. The recombinant plasmids were shown to transfer to C. beijerinckii and were expressed as autonomously replicating vectors. The use of these plasmids as cloning and shuttle vectors for C. beijerinckii is discussed.

Cross-reactivity between RSV-induced tumor antigen and B5 MHC alloantigen in the chicken.

Lymphocytes from chickens homozygous (B2B2) at the major histocompatibility complex (MHC) were tested for cytotoxic activity against five types of target chicken embryo fibroblasts (CEF). Lymphocytes from B2B2 chickens bearing RSV-induced tumors lysed in vitro targets of B2B2 and B5B5 RSV-infected CEF and B5B5 normal CEF, but did not lyse B2B2 and B24B24 normal CEF. Lymphocytes from normal B2B2 chickens did not lyse any of the five types of CEF targets. Alloantisera absorption studies showed that both RSV-infected and uninfected CEF shared alloantigens, in particular B-F alloantigens, with syngeneic erythrocytes. Absorption with B2B2 RSV-infected CEF significantly lowered the titer of B2B2 anti-B5B5 alloantisera. Cross-reactivity between B5 antigen(s) and tumor-associated antigen was suggested and the nature of the cross-reactivity was discussed. It is hypothesized that this cross-reactivity prevents B5B5 chickens from recognizing RSV-induced tumors as foreign, enhances tumor growth and leads to death of the host.

B locus (MHC) effect upon regression of RSV-induced tumors in noninbred chickens

The incidence of regression of Rous sarcoma virus (RSV)-induced tumors in a noninbred line of New Hampshire chickens was approximately six percent (Cotter et al. 1973, Collins et al. unpublished data). But in a mating of one sire with two dams from this line, the mean incidence of tumor regression (of 30 progeny developing tumors) was 50 percent (Collins et al. unpublished data). Thus, although essentially a progressor line, certain individuals transmitted genes favorable to tumor regression. More recently Collins and co-workers (1977) studied the fate of RSVinduced tumors in an F 2 population from a cross of RPRL inbred lines 61 and 151 which was segregating at three alloantigen loci(B, D and I). Among the F 2 segregants B:B 2, B:B 5, and BSB 5 the percentage of chickens dying of terminal tumors by 70 days post inoculation was 5, 26 and 93, respectively. Seventy-five percent of B:B 2 chickens completely or partially regressed their tumors in contrast to none of the BSB 5 animals. The D and I loci had no detectable effect on tumor regression. Schiermann and co-workers (1977) concluded that regression of RSV-induced tumors was a dominantly inherited trait controlled by a gene within, or closely linked to, the major histocompatibility complex (MHC). Thus, in the chicken the MHC, for which the B blood group system serves as a marker, has a gene(s) which dramatically affects the fate of RSV-induced tumors. The objective of this research was to determine the effect of genes in the immediate chromosomal region of the MHC on tumor regression in this line of New Hampshires. Chickens were produced in two hatches from a flock mating of a noninbred line of New Hampshires, U N H 105, known to be genetically susceptible to subgroup A RSV. Chicks were blood typed for B alloantigens at 4 weeks of age using reagents developed in stocks originating from inter se matings of crosses between White Leghorns and experimental birds of heavy breed origin. Three alleles, B 23, B 24 and B 26 had been previously established as existing in this line (Brilles, unpublished). Using appropriate reagents, the 131 chicks from the two hatches were classified into six genotypes-B23B 23, B24B 24, B26B 26, B23B24, B2SB 26, and B24B26. A highly purified pseudotype of Bryan high-titer Rous sarcoma virus designated BH RSV(RAV-1),

Increased growth of RSV-induced tumors in chickens partially tolerant to MHC alloantigens

Chickens withB2B2 MHC genotypes were made partially tolerant to B5 MHC cell-surface antigens and the fate of their Rous-sarcoma-virus (RSV)-induced tumors was determined.B2B2 chickens partially tolerant to viable or lysed white blood cells (WBC) or viable red blood cells (RBC) fromB5B5 chickens had a significantly higher incidence of tumor progression than untreated, PBS-treated, orB2B2 chickens inoculated with WBC from otherB2B2 chickens. The criteria for tolerance were absence of antibody titer to the cell type inoculated and acceptance of allografts fromB5B5 donors byB2B2 chickens. Graft-vs-host reactions occurred only inB2B2 chickens inoculated with viable WBC fromB5B5 chickens. It appears thatB2B2 chickens partially tolerant to B5 antigens failed to mount a successful immune response to RSV-induced tumors partly because a B5 MHC antigen(s) cross-reacted with a tumor associated antigen(s) thereby severely limitingB2B2 host recognition of the tumor as foreign. Since WBC and RBC cell-surface antigens appear to contribute similarly to the effect, theB-F- region of the MHC may be involved.

Protoplast formation, L-colony growth, and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

SummaryProtocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.

Characterization ofBacillus subtilis phage 41c

Bacillus subtilis phages 41c and SPPI were compared. They were identical in their morphologies, their patterns of infectivity, and the buoyant densities of native and denatured DNA. However, differences in plaque morphologies, protein subunit molecular weights, and cleavage patterns generated by treatment of each DNA with restriction endonucleases indicated that phage 41c was not identical to SPPI. It is proposed that phage 41c be considered a separate member of the group-5B. subtilis phages, a group currently comprising only phage SPPI.