PHYLOGENIES AND THE ANALYSIS OF EVOLUTIONARY SEQUENCES,WITH EXAMPLES FROM SEED PLANTS

Studies of character evolution have frequently relied on ahistorical correlations rather than on phylogenies. However, correlations do not estimate the number of times that a trait evolved, and they are insensitive to the direction or the temporal sequence of character transformation. In contrast, cladograms can provide this information. A cladistic test of the hypothesis that the evolution of dioecy is favored in animal-dispersed plants indicates that dioecy may have originated somewhat more often in such lineages. Nevertheless, differences in rates of speciation or extinction must largely account for the observed species-level correlation between dispersal and breeding system. In considering the evolution of individual traits, cladograms help identify the context in which a feature evolved and specify which organisms should be compared in evaluating the causes of character change. Determining whether a feature and a performance advantage were strictly historically correlated or followed one another in sequence helps to distinguish whether the trait is an adaptation or an exaptation for the function. For example, cladograms of seed plants suggest that double fertilization arose incidentally prior to the origin of angiosperms and that the resulting product was later co-opted and elaborated as a nutritive tissue for the developing embryo. The order of character assembly in a lineage also bears on the evolution of functional and developmental interdependencies. In particular, it may be possible to trace the evolution of a character's “burden” from an initial period, during which change is more likely, through later stages, wherein successful modification is less likely owing to the evolution of dependent characters. The evolution of vessels and of floral phyllotaxis in angiosperms may exemplify this pattern. Recognition that the likelihood of character transformation may change during the evolution of a group warns against character weighting in phylogenetic analysis.     

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein.

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.     

Discrimination between oral streptococci by pyrolysis gas-liquid chromatography.

Washed organisms, including strains of Streptococcus mitior, S. mutans, and S. sanguis, were examined by curie-point pyrolysis gas-liquid chromatography. A linear discriminant function based upon three items from the output data was adequate for segregating the strains according to species. Strains with intermediate properties were also encountered. Sources of variability in cultures were evaluated, chromatographic performance was maintained throughout the investigation, and matching performance from a duplicate pair of chromatographic columns was demonstrated.     

Replication of colicin E1 plasmid DNA in vivo requires no plasmid-encoded proteins.

A derivative of bacteriophage lambda containing a colicin E1 plasmid replicon was constructed by recombinant DNA techniques. This phage, lambdacol100, has two functional modes of DNA replication; it can replicate via either plasmid or phage replication systems. lambdacol100 has been used to introduce the colicin E1 plasmid replicon into Escherichia coli previously treated with chloramphenicol to block protein synthesis. Under these conditions, lambdacol100 DNA is replicated normally as a colicin E1 plasmid. This suggests that colicin E1 plasmid replication in vivo does not require any plasmid-encoded proteins.     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.     

A generalized method of subcloning DNA fragments by restriction site reconstruction: application to sequencing the amino-terminal coding region of the transforming gene of Gazdar murine sarcoma virus

The technique of restriction site reconstruction was generalized so as to allow the subcloning of any DNA fragment and its subsequent reexcision with EcoRI, XbaI, XhoI or HindIII. After excision, the 3' terminus of each strand will be derived from the starting nucleic acid, permitting the use of such fragments as primers for nucleotide sequencing by primer extension methods. The technique was used to subclone a 56 base pair BstNI-DdeI fragment of Moloney murine sarcoma virus (Mo-MSV) as a unique HindIII-HindIII fragment. This fragment then served as a primer to sequence a portion of the RNA genome of Gazdar murine sarcoma virus (Gz-MSV). The nucleotide sequence which was obtained indicated that the transforming gene of Gz-MSV arose by at least two recombination events involving murine leukemia virus (MLV) and the cellular homologue c-mos. This analysis suggests that a virus indistinguishable from Mo-MSV was an intermediate in the formation of Gz-MSV.