Rat growth hormone gene introns stimulate nucleosome alignment in vitro and in transgenic mice.

Average hepatic expression (mRNA per cell per gene) of a metallothionein-rat growth hormone (rGH) gene with its natural introns was about 15-fold higher than an intronless version when tested in transgenic mice. We examined the idea that intron removal leads to an alteration in chromatin structure that might be responsible for this effect. Using an in vitro chromatin assembly system, we observed that nucleosomes were aligned in a characteristic ordered array over the gene and promoter when all introns were present. Linker histones were necessary for this alignment to occur. In contrast, nucleosome alignment was perturbed in constructs lacking some or all of the introns. A similar disruption of nucleosome alignment was observed when comparing chromatin from livers of transgenic mice carrying rGH transgenes with or without introns. In vitro, sequences at the 3' end of the rGH gene position nucleosomes and facilitate nucleosome alignment upstream; however, nucleosome alignment does not occur on the approximately 3 kb of downstream flanking rat sequence. These observations suggest that signals present in genomic rGH DNA may serve to establish appropriate nucleosome alignment during development and, possibly, to restore nucleosome alignment to the transcribed region after disruption incurred by the passage of an RNA polymerase molecule, thereby facilitating subsequent rounds of transcription.

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.