Atomic-scale observation of temperature and pressure driven preroughening and roughening.

Preroughening and roughening transitions are observed on the GaAs(001) surface using scanning tunneling microscopy. By tuning the substrate temperature or As4 pressure the surface morphology can be made free of islands, covered with one monolayer high islands or covered with islands on top of islands forming a wedding-cake-type structure. These three distinct surface morphologies are classified as ordered flat (OF), disordered flat (DOF), and rough within the restricted solid-on-solid model. Here, the DOF phase is macroscopically flat; however, an up-down-up-down step pattern persists across the entire surface. Using this model we have determined the next-nearest-neighbor interaction energy to be about 0.05 eV.

Spatially resolved spin-injection probability for gallium arsenide.

We report a large spin-polarized current injection from a ferromagnetic metal into a nonferromagnetic semiconductor, at a temperature of 100 Kelvin. The modification of the spin-injection process by a nanoscale step edge was observed. On flat gallium arsenide [GaAs(110)] terraces, the injection efficiency was 92%, whereas in a 10-nanometer-wide region around a [111]-oriented step the injection efficiency is reduced by a factor of 6. Alternatively, the spin-relaxation lifetime was reduced by a factor of 12. This reduction is associated with the metallic nature of the step edge. This study advances the realization of using both the charge and spin of the electron in future semiconductor devices.

Association between alleles of the ovine major histocompatibility complex and resistance to footrot.

Variation in natural resistance to footrot may be genetically derived, implying that genetic markers for resistance may exist and allow selection of superior animals. In this study association between variation within the ovine MHC class II region and resistance to footrot was investigated in two trials. Half-sib progeny were subjected to a field challenge with footrot and their condition subsequently recorded. The animals were then typed at their MHC class II loci to investigate associations between inherited paternal haplotype and footrot status. In the first trial an association between MHC haplotype and footrot status was observed across all animals (P = 0.005), when the self-curing and resistant animals were combined (P = 0.002) and when the self-curing animals were excluded from the analysis (P = 0.001). No association was observed in the second trial, a result attributed to the dry weather conditions which led to poor disease transmission and unreliable disease classification.

Polymorphism at the ovine major histocompatibility complex class II loci.

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) (MhcOvar) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQA2 had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.

Expression of human Krev-1 gene in lungs of transgenic mice and subsequent reduction in multiplicity of ethyl carbamate-induced lung adenomas.

Mice of the A/J strain are useful models of lung cancer because they develop tumors spontaneously or after treatment with ethyl carbamate. These tumors are thought to arise from either Clara cells (papillary tumors) or alveolar type 2 cells (alveolar tumors); like many human lung adenocarcinomas, the mouse tumors involve Kiras activation. Transformation with Ki-ras can be reversed by coexpression of the Krev-1 gene in tissue culture. To test the tumor suppressor activity of Krev-1 in vivo, we produced transgenic A/J mice expressing Krev-1 under the control of the rabbit uteroglobin promoter, which directs expression of heterologous genes to the lung Clara cells. Krev-1 was expressed specifically in the lungs of transgenic mice. Sixty-six mice (35 transgenic and 31 nontransgenic) from three lines were given ethyl carbamate, and the numbers of resulting lung tumors were compared between transgenic and nontransgenic animals. The mean number (+/-standard deviation) of ethyl carbamate-induced lung tumors was 21.7 +/- 1.3 in transgenic mice and 26.9 +/- 1.3 in their nontransgenic littermates (P < 0.01). Sequencing of polymerase chain reaction-amplified ras DNA from 15 transgenic mouse tumors and 16 nontransgenic mouse tumors (controls) detected mutations in codon 61 in 13 tumors from the transgenic group and 11 tumors in the control group, whereas mutations in codon 12 were detected in only one tumor in the transgenic group and in four tumors in the controls. Together, these data demonstrate for the first time the tumor suppressor activity of Krev-1 in vivo and suggest that Krev-1 tumor suppressor activity may be specific for cells harboring mutations in codon 12 of ras.

Improved wool production in transgenic sheep expressing insulin-like growth factor 1.

Transgenic sheep were produced by pronuclear microinjection with a mouse ultra-high-sulfur keratin promoter linked to an ovine insulin-like growth factor 1 (IGF1) cDNA. Five transgenic lambs resulted from the microinjection of 591 embryos; one male and one female showed IGF1 expression in the skin. A progeny test of the ram was carried out by matings to 43 non-transgenic ewes. Of 85 lambs born, 43 (50.6%) were transgenic. At yearling shearing (approximately 14 months of age), clean fleece weight was on average 6.2% greater in transgenic animals than in their non-transgenic half-sibs, with a greater effect in males (9.2%) than females (3.4%). Transgenics showed a small but significant increase in bulk, but male transgenics had a lower staple strength than female transgenics and non-transgenics which did not differ significantly. There were no significant differences in fiber diameter, medullation, and hogget body weight. To our knowledge this is the first reported improvement in a production trait by genetic engineering of a farm animal without adverse effects on health or reproduction.

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

Inhibition, tissue distribution and hormonal specificity of a promoter-binding factor for the uteroglobin gene.

We had earlier reported a uteroglobin promoter-binding factor in nuclei from progesterone-stimulated rabbit endometrium that was inhibited by a factor in nuclei without progesterone stimulation or from non-target tissues (Rider, V., and Bullock, D.W., 1988. Biochem. Biophys. Res. Comm. 156, 1368-1375). In the course of purification of the inhibitory activity, the effect was shown to be due to contaminating genomic DNA. The inhibitor was destroyed by treatment with DNase I and resisted phenol-chloroform extraction. Fractionation of nuclear extracts on columns of DEAE-Sepharose separated the inhibitor and revealed the presence of binding activity in unstimulated or estrogen-treated endometrium, as well as in liver, lung and ovary. The tissue and hormonal specificity of the promoter binding factor is thus less restricted than recently reported.

Geographical origin of an introduced insect pest, Listronotus bonariensis (Kuschel), determined by RAPD analysis.

The Argentine stem weevil, Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae), is an important introduced pasture pest in New Zealand. In this study geographical populations of this species were analysed using polymerase chain reaction-based randomly amplified polymorphic DNA (RAPD), in an attempt to determine the geographical origin of the pest. Morphologically indistinguishable individuals were collected from nine South American, five New Zealand and one Australian populations. Ten primers were screened for usefulness, two of which revealed significant, scorable polymorphisms between these populations. The results indicated that the sampled New Zealand L. bonariensis populations originated from the east coast of South America.

Chromosomal position effects and the modulation of transgene expression.

Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.

A simple two-step method for efficient blunt-end ligation of DNA fragments.

The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration). Standard protocols recommend an average DNA concentration at which both events may occur. Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised. We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight. The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols. Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.

Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

Expression and regulation of the rabbit uteroglobin gene in transgenic mice.

The rabbit uteroglobin (UG) gene, with varying lengths of 5' flanking sequence, was introduced into the mouse genome to investigate the DNA sequences required for tissue-specific expression and regulation by steroid hormones. The pattern of expression and steroid hormone regulation of the transgene was compared to the expression and regulation of the endogenous mouse UG-like gene. In the rabbit, UG is induced in the uterus by progesterone and is expressed constitutively in the lungs, where it is weakly regulated by glucocorticoids. Genomic DNA fragments containing the complete UG-coding sequence with 4.0 (UG4.0), 3.0 (UG3.0), 2.3 (UG2.3), or 0.6 (UG0.6) kilobases of 5' flanking sequence were used to establish lines of transgenic mice. Expression of UG mRNA was observed in the lungs of UG4.0 (2/4 lines), UG3.0 (4/4 lines), UG2.3 (1/2 lines), and UG0.6 (4/4 lines) mice. Uterine expression was observed in UG3.0 (3/4 lines), UG2.3 (1/2 lines), and UG0.6 (2/4 lines). In the lungs of UG3.0 and UG2.3 mice, RNA expression was stimulated by treatment with dexamethasone. In the one line of UG3.0 mice examined, UG was regulated by ovarian steroids in the uterus. The endogenous mouse UG-like gene showed the major site of expression to be in the lung. Unlike the transgene, the endogenous gene was more strongly stimulated by glucocorticoids. Thus, we conclude that the cis elements needed for pulmonary expression of UG are contained within the UG2.3 fragment used to generate transgenic mice, but that other elements are required for full glucocorticoid regulation. Also, the transgene did not show the full uterine expression observed in the rabbit, but regulation by the ovarian steroids was observed.

Effect of passive immunization against progesterone on its distribution in circulation and tissues of mice.

Anti-progesterone monoclonal antibody, injected into mice 32 h after mating at a dose that blocks the establishment of pregnancy, produced a significant reduction in the concentration of progesterone in the ovary and uterus within 6 h after treatment. Uterine concentrations remained lower in treated compared with control animals for at least 24 h after injection. There was an associated transient increase in plasma LH and FSH concentrations, but there was no change in plasma prolactin values. The percentage of total progesterone in the circulation that was unbound was reduced after treatment, but the concentration of unbound progesterone was increased. Studies of antibody binding of steroid in the presence of uterine progesterone receptor protein showed that there was a stoichiometric relationship in the distribution of ligand between the two binders. The present findings suggest that the effects of passive immunization against progesterone are associated with perturbation of tissue concentrations of steroid in the target organ as a result of high antibody concentrations in the circulation.

Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.

Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.

Servomechanism of prolactin and progesterone in regulating uterine gene expression.

To investigate the interaction of PRL and progesterone in regulating uterine gene expression, we have quantitated the concentration of PRL receptor and of uteroglobin (UG) mRNA in the endometrium of rabbits of different ages and after treatment with different hormones. During uterine differentiation in 2- to 4-week old rabbits, a marked increase in unoccupied uterine PRL receptor number was observed, presumably increasing uterine sensitivity to PRL. Receptor values for 4-week old rabbits were comparable to values for sexually mature, estrous females, but were lower than in 5-day pseudopregnant (PSP) animals. When total PRL receptor was determined by Scatchard analysis after in vitro desaturation with MgCl2, PSP animals again expressed the highest receptor concentration with no changes in the dissociation constant (Kd) values. To determine whether progesterone regulates uterine PRL receptor, long term ovariectomized rabbits (greater than 12 weeks) were treated with various combinations of hormones, and unoccupied and total uterine PRL receptors were determined. Progesterone treatment resulted in the highest concentration of both unoccupied and total PRL receptor after desaturation and removal of anti-ovine PRL antibodies with MgCl2. The value for total uterine PRL receptor was equivalent to the value for mammary gland, and the Kd values (2-4 x 10(-10) M) were similar. Treatment of long term ovariectomized rabbits with progesterone, with or without estradiol, produced an increase (P less than 0.05) in the UG mRNA content, which also occurred in PSP animals. PRL alone had no effect on UG mRNA but PRL plus progesterone increased (P less than 0.05) UG mRNA in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone-dependent binding of a trans-acting factor to the uteroglobin promoter.

A trans-acting factor that specifically binds to the uteroglobin (UG) gene promoter has been identified. Binding activity was absent in non-target tissues (lung, liver) in HeLa cell nuclear extracts, and in target tissue (endometrium) in the absence of progesterone. Mixing experiments revealed an inhibitor of promoter binding in the absence of progesterone and in nonspecific nuclear extracts. Inhibition of binding in the endometrium was reversed by the action of progesterone. The results suggest that binding of a transcription regulatory factor to the UG promoter switches from negative to positive with the action of progesterone. The binding activity corresponds to the expression of the UG gene and this protein may be, therefore, a coordinately regulated trans-acting factor which regulates UG in a tissue-specific manner.