Sieve cells in Phloem of a middle devonian progymnosperm.

Phloem tissue from a Middle Devonian member of the Aneurophytales (Progymnospermopsida) is described. This may be the oldest firm evidence of conducting elements of the phloem, extending our knowledge of this tissue back some 35 million years. The discovery indicates a close phylogenetic relation between progymnosperms and gymnosperms and provides a basis for investigating patterns of specialization in the phloem of these groups of plants.

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Glycine 119 of bovine growth hormone is critical for growth-promoting activity.

Bovine GH (bGH) analogs with single amino acid substitutions at positions 117 (bGH-E117L), 119 (bGH-G119R), and 122 (bGH-A122D) were generated. These analogs bind to mouse liver membrane preparations with affinities similar to native bGH. However, transgenic mice which express the analogs demonstrate different phenotypes ranging from dwarfism to gigantism. For example, expression of bGH or bGH-E117L result in large transgenic mice. In contrast, transgenic mice with a growth phenotype similar to nontransgenic animals result from expression of bGH-A122D. Surprisingly, transgenic mice with relatively high serum levels of bGH-G119R possessed a dwarf phenotype. Together these results suggest that Gly 119 and Ala 122 are involved in growth-promoting activity of GH.

Amino acid residues in the third alpha-helix of growth hormone involved in growth promoting activity.

The third amphiphilic alpha-helix of GH has been found to be an important motif in the biological activities of the molecule. To further characterize this growth-promoting domain, three bovine (b) GH analogs were designed: one contained a scrambled third amphiphilic alpha-helix (SAH); a second contained a scrambled hydrophilic region of the helix (SAP); and a third contained a scrambled hydrophobic region of the helix (SAB). Transgenic mice that expressed these mutated bGH genes were produced. SAH transgenic mice displayed a phenotype identical to nontransgenic littermates. SAB transgenic mice grew slightly larger than nontransgenic littermates but remained smaller than bGH transgenic mice. On the other hand, SAP transgenic mice exhibited a dwarf phenotype. We subsequently generated individual amino acid substitutions in the hydrophilic region of the helix. The results from the growth rates of corresponding transgenic mice demonstrated that most bGH analogs with individual amino acid substitution within the third alpha-helix retained wild type-like growth-promoting activity except those with alterations at positions 115, 119, 122, and 123. Together these residues are predicted to form a cleft in the helix. To further substantiate the importance of the cleft, we deleted Gly 119 (delta 119). This resulting bGH analog was inactive in vivo as well as in in vitro assays. These results indicated that the primary structure of the third alpha-helix is critical for GH's growth-promoting activity and Gly 119 is a crucial amino acid in this region. Three adjacent amino acids, Asp 115, Ala 122, and Leu 123, also contribute to the growth-enhancing ability of the molecule.

Transgenic mice: a decade of progress in technology and research.

In little more than a decade, the techniques developed for altering the genetic makeup of laboratory and livestock animals and plants have changed the landscape of biological research. It is now possible to introduce virtually any cloned gene into the germ line and study the expression pattern and effects of the introduced gene, or transgene. This has allowed the extension of in vitro and in vivo cell-culture studies into whole animal systems in which the introduced gene is subject to all normal regulatory processes from the onset of development. Although there have been reports of foreign gene expression resulting from direct injection of DNA in animals (e.g., Wolff et al., 1990; Zhu et al., 1993), transgenic animals are the primary model system for examining molecular genetic phenomena in vivo.

Intragenic sequences are required for cell type-specific and injury-induced expression of the rat peripherin gene.

Peripherin is a 57 kDa type III intermediate-filament protein that is thought to play a role in axonogenesis both during development and following nerve injury (Oblinger et al., 1989; Escurat et al., 1990; Gorham et al., 1990; Troy et al., 1990b). We have used transgenic mouse technology to define peripherin gene sequences that are necessary for cell type-specific expression and for the increase in peripherin that occurs in response to axonal injury. Correct temporal and nervous system-specific expression resulted when 5.8 kilobases of peripherin 5' flanking sequence were linked to a reporter gene, but precise cell type-specific expression was achieved only when intragenic sequences were included. When intragenic sequences were present, peripherin transgenes were expressed in dorsal root ganglion neurons and spinal cord motor neurons and were upregulated in these cells following nerve injury.

Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.

To determine the importance of the third alpha-helix in bovine growth hormone (bGH) relative to growth-related biological activities, the following experimental approach was used: (i) mutagenesis of helix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies of wild-type and mutated bGH; and (iv) expression of the mutated gene in the transgenic mouse. An altered bGH gene (pBGH10 delta 6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGH10 delta 6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growth-suppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule.

Mutations in the third alpha-helix of bovine growth hormone dramatically affect its intracellular distribution in vitro and growth enhancement in transgenic mice.

To investigate the relationship between the secondary structure of the third alpha-helix (amino acids 109-126) of bovine growth hormone (bGH) and the biological activity of the molecule, proline or glycine residues have been used as substitutes for native amino acids at positions 114, 118, 121, and 126, respectively. Mutations at the positions 114, 118, and 121 resulted in a dramatic decrease in bGH secretion by transiently transfected mouse L cells whereas the substitution of glycine for glutamate at position 126 (bGH-E126G) did not affect secretion. Immunofluorescence staining revealed that those nonsecretory bGH mutations possessed a different intracellular location as compared with wild-type bGH or the mutated secretory forms of bGH. Similar results were seen in the distribution of these mutated bGH molecules in transfected rat GH-3 cells. Transgenic mice that express wild-type bGH or bGH-E126G grew to approximately 1.6 times the mass of nontransgenic littermates. Transgenic mice that express two nonsecretory forms of mutated bGHs were found to lack the enhanced mouse growth phenotype in spite of elevated levels of serum bGH. These results suggest that the secondary structure in the third alpha-helix of bGH may be important for efficient intracellular targeting in vitro and in growth promotion in transgenic mice.

FLP-mediated site-specific recombination in microinjected murine zygotes.

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous systems (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occurred at the one-cell stage and deleted a chromosomal tandem array of a FRT containing lacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.

The human growth hormone transgene: expression in hemizygous and homozygous mice.

Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5-fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.

Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS alpha overexpression.

To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 +/- 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF) was not different from that of control mice at baseline (LVFS, 40 +/- 3% versus 36 +/- 2%; LVEF, 78 +/- 3% versus 74 +/- 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 micrograms/kg per minute) infusion were higher than the values in control mice (LVFS, 68 +/- 4% versus 48 +/- 3%; LVEF, 96 +/- 1% versus 86 +/- 3%; P < .05). Norepinephrine (NE, 0.2 micrograms/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 +/- 4% versus 47 +/- 3%; LVEF, 93 +/- 2% versus 85 +/- 3%; P < .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 +/- 2% with ISO (0.02 micrograms/kg per minute) and by only 13 +/- 2% in three wild-type control mice (P < .01). NE (0.1 micrograms/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 +/ 8% versus 28 +/- 4%, P < .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 +/- 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 +/- 1.4% versus 1.2 +/- 0.3%, P < .05) and in the cross-sectional area of myocytes (298 +/- 29 versus 187 +/- 12 micron2, P < .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs-adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.

Cardiomyopathy induced by cardiac Gs alpha overexpression.

The goal of this study was to determine whether chronic endogenous sympathetic stimulation resulting from the overexpression of cardiac stimulatory G protein alpha subunit (Gs alpha) in transgenic mice (15.3 +/- 0.1 mo old) resulted in a clinical picture of cardiomyopathy. The left ventricular ejection fraction, measured by echocardiography, was reduced in older mice with Gs alpha overexpression (50.4 +/- 5.4%) compared with age-matched control mice (70.9 +/- 1.6%; P < 0.05). When ejection fractions were compared at similar heart rates, the Gs alpha mice exhibited a greater left ventricular end-diastolic dimension than control mice (4.3 +/- 0.2 vs. 3.7 +/- 0.1 mm; P < 0.05). Baseline heart rates were elevated in conscious Gs alpha mice (722 +/- 27 beats/min; n = 5) compared with control mice (656 +/- 28 beats/min; n = 5). Moreover, electrocardiographic monitoring demonstrated a high incidence of arrhythmias. Increased mortality compared with control mice (31.6 vs. 3.0%; P < 0.01) was also observed. Thus older mice with Gs alpha overexpression exhibit many of the features of dilated cardiomyopathy. This study supports the concept that chronic sympathetic stimulation over an extended period of time, i.e., over the life of an animal, is deleterious and actually may result in cardiomyopathy.