Expression of new members of the prolactin growth hormone gene family in bovine placenta. Isolation and characterization of two prolactin-like cDNA clones

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone. The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively. bPLP-I, bPLP-II, placental lactogens, prolactins (PRLs), and other prolactin-like proteins isolated from cow, mouse, and rat share 7 common amino acid residues. Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.     

Structural assembly of two-domain proteins by rigid-body docking

Background  

Modelling proteins with multiple domains is one of the central challenges in Structural Biology. Although homology modelling has successfully been applied for prediction of protein structures, very often domain-domain interactions cannot be inferred from the structures of homologues and their prediction requiresab initiomethods. Here we present a new structural prediction approach for modelling two-domain proteins based on rigid-body domain-domain docking.     

Primary ciliary dyskinesia (Siewert's / Kartagener's Syndrome): Respiratory symptoms and psycho-social impact

Background

Although the pathophysiological defect in primary ciliary dyskinesia (PCD; Siewert's / Kartagener's syndrome) is now well characterised, there are few studies of the impact of the condition upon health function, particularly in later life. This study assesses the health impact of the condition in a large group of patients. In addition, it assesses the similarity in age of diagnosis, symptoms and problems of those with situs inversus (PCD-SI) and those with situs solitus (PCD-SS).

Methods

Postal questionnaire sent to members of the UK Primary Ciliary Dyskinesia Family Support Group. The questionnaire contained the St. George's Respiratory Questionnaire (SGRQ) and the SF-36 questionnaire for assessing health status.

Results

93 questionnaires were returned, representing a 66% response rate. Replies were received from similar numbers of PCD-SI and PCD-SS. Individuals with PCD-SI did not show a significant tendency to be diagnosed earlier, and neither did they show any difference in their symptoms, or the relationship of symptoms to age. Respiratory symptoms were fairly constant up until the age of about 25, after which there was a slow increase in symptoms, and a decline in health status, patients over the age of 40 being about one and a half standard deviations below the mean on the physical component score of the PCS. Patients diagnosed earlier in life, and hence who had received more treatment for their condition, had better scores on the SGRQ Impact and Activity scores.

Conclusions

PCD is a chronic condition which has a progressively greater impact on health in the second half of life, producing significant morbidity and restriction of life style. Early diagnosis, and hence earlier treatment, may improve symptoms and the impact of the condition.     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

The DT40 web site: sampling and connecting the genes of a B cell line

Thousands of new vertebrate genes have been discovered and genetic systems are needed to address their functions at the cellular level. The chicken B cell line DT40 allows efficient gene disruptions due to its high homologous recombination activity. However, cloning the gene of interest is often cumbersome, since relatively few chicken cDNA sequences are present in the public databases. In addition, the accumulation of multiple mutations within the same cell clone is limited by the consumption of one drug-resistance marker for each transfection. Here, we present the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html), which includes a comprehensive database of chicken bursal ESTs to identify disruption candidate genes and recyclable marker cassettes based on the loxP system. These freely available resources greatly facilitate the analysis of genes and genetic networks.     

Space–time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the <Emphasis Type="Italic">IAA19</Emphasis> promoter fusion with a destabilized luciferase reporter

Imaging analysis was carried out during the gravitropic response of etiolated Arabidopsis hypocotyls, using an IAA19 promoter fusion of destabilized luciferase as a probe. From the bright-field images we obtained the local deflection angle to the vertical, A, local curvature, C, and the partial derivative of C with respect to time, \(\partial C/\partial t\). These were determined every 19.9 µm along the curvilinear length of the hypocotyl, at ~10 min intervals over a period of ~6 h after turning hypocotyls through 90° to the horizontal. Similarly from the luminescence images we measured the luminescence intensity of the convex and concave flanks of the hypocotyl as well as along the median of the hypocotyl, to determine differential expression of auxin-inducible IAA19. Comparison of these parameters as a function of time and curvilinear length shows that the gravitropic response is composed of three successive elements: the first and second curving responses and a decurving response (autostraightening). The maximum of the first curving response occurs when A is 76° along the entire length of the hypocotyl, suggesting that A is the sole determinant in this response; in contrast, the decurving response is a function of both A and C, as predicted by the newly-proposed graviproprioception model (Bastien et al., Proc Natl Acad Sci USA 110:755–760, 2013). Further, differential expression of IAA19, with higher expression in the convex flank, is observed at A?=?44°, and follows the Sachs’ sine law. This also suggests that IAA19 is not involved in the first curving response. In summary, the gravitropic response of Arabidopsis hypocotyls consists of multiple elements that are each determined by separate principles.     

An agravitropic mutant of Arabidopsis, endodermal-amyloplast less 1, that lacks amyloplasts in hypocotyl endodermal cell layer

We have isolated a new recessive mutant of Arabidopsis thaliana for gravitropism, endodermal-amyloplast less 1 (eal1). eal1 shows reduced gravitropism in hypocotyl, and completely lacks gravitropism in inflorescence stems; root gravitropism is not affected. Starch staining with I-KI solution reveals almost no amyloplasts in eal1 hypocotyls when grown on a sucrose-free medium, though the root columella cells contain as many amyloplasts as wild type. On a medium containing 1% sucrose, eal1 hypocotyls contain as many starch granules as those of wild type, suggesting that starch synthesis is not affected in eal1. The endodermal cell layer which is thought to function as statocytes in hypocotyls is present in eal1. These results suggest that differentiation or development of gravity-responsive amyloplasts are affected in eal1 hypocotyls.     

The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf.

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 microM were studied, with maximum IAA-induced curvature at 100 microM. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg 1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which it effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.