Cloning and sequencing of the cDNA for ovine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)

Abstract

Summary Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants. By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages. The PCR product was cloned into pUC 119, and electroporated into Escherichia coli. The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined. At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively.

Endotoxin-induced maturation of monocytes in preterm fetal sheep lung

“…Sheep GM-CSF was cloned into pGEM-T vector (Promega, Madison WI) from endotoxin-exposed fetal sheep lung cDNA by PCR using primers complementary to the published sheep GM-CSF sequence (NCBI accession no. X 55991) (35). Identity of ovine GM-CSF was confirmed by double-stranded sequence comparison with the published sequence.…”

Molecular cloning, sequencing and structural studies of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) from Indian water buffalo (Bubalus bubalis)

“…Cloning and characterization of bovine, murine, ovine and human GM‐CSF cDNA have already been reported (Gough et al ., ; Wong et al ., ; Leong et al ., ; O'Brien et al ., ). Although the identities between these genes are relatively high, the biological activities of these proteins are generally species specific in their actions, the exception being human GM‐CSF (hGM‐CSF), which has limited activity on bovine bone marrow cells (Cantrell et al ., ; Leong et al ., ).…”

Influence of Hypoxia and Glucose Deprivation on Tumour Necrosis Factor-Alpha and Granulocyte- Macrophage Colony-Stimulating Factor Expression in Human Cultured Monocytes

“…RNA (10 Ìg, or the maximum amount available from the extraction) was separated on a 1.2% formaldehyde denaturing gel and transferred to Hybond N + nylon membranes (Amersham, UK) by alkali blotting (0.05 M sodium hydroxide). GM-CSF mRNA was detected by Northern hybridisation (Megaprime Labelling Kit, Amersham, UK) using a 450-bp ovine cDNA probe [31] labelled with ·-32 P-dCTP (Amersham, UK; n = 9 for normoxia/hypoxia + glucose +/-LPS; n = 5 for normoxia/hypoxia -glucose +/-LPS). The membranes were hybridized overnight at 65°C and washed once with 5 !…”