Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.

We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells. As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P. pastoris.

A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts.

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR) performed at 42 degrees C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.

Thymocyte reconstitution of athymic and athymic-asplenic mice: graft rejection and antibody synthesis.

Hereditarily athymic and athymic-asplenic mice were reconstituted with syngeneic thymocytes at various times prior to and after tumor challenge. These mice were able to reject heterotransplanted human tumors in as little as 19 days after thymocyte transfer but were unable to do so when the thymocytes were given 7 days after tumor implantation. The humoral immunity of athymic nude mice was fully restored as determined by the primary immune response to sheep erythrocytes (SRBC), a thymocyte-dependent antigen. Athymic-asplenic mice failed to respond to SRBC after reconstitution and this was attributed to the absence of the spleen in these mice.

Reproducible metastatic growth of K-562 human myelogenous leukemia cells in nude mice.

A reproducible metastatic growth of K-562 human myelogenous leukemia cells occurred in immunodeficient athymic (nude) mice. Although previous studies have shown that K-562 cells grow as local subcutaneous myelosarcomas which continuously release leukemia cells into the systemic circulation in adult mice, metastases were not observed. However, the subcutaneous "priming" of newborn nude mice resulted in the metastatic proliferation of leukemia cells in the lungs, kidneys, brain, and lymph nodes. Three sc injections of 5 X 10(6) K-562 cells on days 1, 7, and 14 of life produced metastases in 51% of the mice. When the initial series of injections was followed by iv injections on days 35 and 42, the incidence of metastases increased to 67%. Karyotypes demonstrated that the tumor cells retained the same human chromosome markers as those in the human patient and tissue culture. These procedures may provide a model for study of the mechanisms of metastases and for chemotherapeutic and immunotherapeutic trials against metastases of neoplasms of human origin.

High incidence of mammary tumors in mice with inherited asplenia carriers for the nude gene.

A colony of mice suffering from dominant hemimelia associated with agenesis of the spleen has been developed and characterized during the past 7 years. The hereditarily asplenic (Dh/+) mice have a very low incidence (9%) of spontaneous mammary tumors (SMT). Asplenic (Dh/+) females were mated with mice homozygous (nu/nu) for hereditary athymia (nude) having a BALB/c background. BALB/c females heterozygous for the nu gene and with spleen (nu/+,+/+) have a moderate incidence (12%) of SMT, whereas nu/+,Dh/+ breeders have a drastic increase in the incidence of SMT to 46% when bred under identical conditions. Since all parent strains have a very low incidence of SMT, it appears that the spleen agenesis is a major factor accounting for an earlier and higher incidence of SMT in hereditarily asplenic (nu/+,Dh/+) mice than in normal (nu/+,+/+) siblings. The SMT express mammary tumor virus antigen(s) and possess estrogen, progesterone, and glucocorticoid receptors. The SMT rapidly metastasize and kill the host within 30 to 45 days. The BALB/c asplenic mice with SMT represent a unique model relevant to human breast cancer and for study of the function of the spleen in the development of solid tumors in general and of SMT in particular.

Heterotransplantation of human neoplasms.

The heterotransplantation of 39 solid human neoplasms into athymic and asplenic-athymic mice resulted in the successful growth of 28 of the transplants. However, only 17 of the tumors could be maintained in serial passage. Metastasis or local infiltration by the transplanted tumors was not observed. Melanomas and colorectal carcinomas grew well whereas other types grew poorly or not at all. Possible factors responsible for the poor growth or lack of growth are discussed. The rapid growth of a melanoma in newborn mice in contrast with the slow growth of the same tumor in adult mice suggests a new experimental approach for transplantation of malignancies which previously have been difficult to establish.

Spontaneous fibrosarcoma in a thymus-transplanted nude mouse.

A fibrosarcoma was observed in a 6-month-old nude mouse that had a neonatal thymus transplant and had been kept since birth under nearly pathogen-free conditions. This was the first tumor found in more than 1,000 nude mice maintained in a colony in which mammary carcinomas were observed among the female heterozygous breeders. This finding confirmed the fact that malignant neoplasms are rare in an animal model that lacks T-cell-mediated immunologic capability and apparently contradicted the postulates of the immunosurveillance theory.