Cloning of a high growth gene

#The present invention generally relates to modifying growth in domestic animals, and more particularly to oligonucleotide probes useful to isolate genomic clones so as to improve growth performance and efficiency in domestic animals, such as by knocking out loci related to the control of growth or utilizing identified growth quantitative trait loci in marker-assisted selection programs with domestic animals.

#In domestic animals, many different hormones (e.g. growth hormone, sex steroids) are known to have an important function in maintaining normal animal growth and to be effective growth promoters when administered exogenously (e.g. Kopchick, Livestock Prod. Sci., 27:66-75, 1991). Some transgenic animals have been caused to express a growth hormone, and increased growth of such transgenic animals has been reported. Palmiter et al. (Nature, 300:611, 1982) microinjected the male pronucleus of fertilized mouse eggs with a DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone. Several of the transgenic mice developed from the genetically modified zygote exhibited a growth rate substantially higher than that of control mice. Palmiter et al. (Science, 222:809, 1983) demonstrated that a similar enhancement of growth could be obtained in transgenic mice bearing an expressible human growth hormone gene. A like effect is observed when human growth hormone releasing factor is expressed in transgenic mice. (Hammer et al., Nature, 315:413, 1985). Bovine growth hormone has also been expressed in transgenic animals (McGrane et al., J. Biol. Chem., 263:1144351, 1988; Kopchick et al., Brazil. J. Genetics, 12:37-54, 1989).

#U.S. Pat. No. 5,350,836, inventors Kopchick et al., issued Sep. 27, 1994, entitled “Growth Hormone Antagonists,” describes administration of a mutein that has growth-inhibitory activity in vertebrates and may be administered to mammals, such as bovines, when growth inhibition is desirable. Alternatively, a gene coding for the hormone is suggested for introduction into a prenatal form of a mammal to produce growth-inhibited animals.

#FIG. 1 illustrates a physical map of the high growth (“hg”) region where: (A) are polymerase chain reaction (PCR)-based markers (sequence tagged sites, STSs) from ends of clones shown non-italicized, the genetic (microsatellite) marker “D10Mit69” is italicized, and a PCR-based marker derived from an exon trapping product we hereinafter call “B308A” is typed in bold; (B) are genomic DNA of control mice tested as progenitors of high growth mice, AKR/J, C3H/HeJ, C57BL/6J, and DBA/2J, with the high growth mouse line being tested being C57BL/6J-hghg; (C) are Yeast Artificial Chromosome (YAC) clones, and (D) are Bacterial Artificial Chromosome (BAC) clones;

#FIG. 2 illustrates Northern blots with hybridization to mouse embryonic stages and adult mouse tissues using the candidate exon “B308A” as a probe (two upper panels), and as a control the bottom two panels are blots stripped off the “B308A” probe and reprobed with cDNA for human B-actin gene;

#FIG. 3A is the nucleotide sequence of the cDNA in the inventive mouse clone called “B308A-6-1” (SEQ ID NO:1);

#SEQ ID NO:1 illustrates a mouse cDNA, which is the first and only candidate gene in the high growth (“hg”) region believed to be discovered to date. The hg region appears to be highly conserved, as will be more fully described herein.

#Turning to FIG. 1, the deleted microsatellite marker, D10Mit69, was utilized as an entry point to physical cloning of the hg-containing segment using Yeast Artificial Chromosome (YAC) and Bacterial Artificial Chromosome (BAC) cloning systems. The size of the deletion in high growth mice, estimated from the clone lengths, is on the order of a half million base pairs.

#The open reading frame of the mouse B308A-6-1 (FIG. 3A) is predicted to encode 199 amino acids (FIG. 3B) which share very high homology (178/199 identical amino acids) with the human protein. The nucleotide sequence of the original exon-trap clone, with the position of primers 1, 2, 3, and 4 is indicated by FIG. 4 (this partial sequence is SEQ ID NO:2). The bovine sequence, which is yet a partial coding sequence, is SEQ ID NO:3 and is shown by FIG. 5. This PCR amplified sequence was from reverse transcribed lactating mammary gland mRNA using the mouse primers 2 and 3 (the primers indicated by FIG. 4).