Michael A. Cantrell

Loss of LINE-1 activity in the megabats.

LINE-1 (L1) retrotransposons are the most abundant type of mammalian retroelement. They have profound effects on genome plasticity and have been proposed to fulfill essential host functions, yet it remains unclear where they lie on the spectrum from parasitism to mutualism. Their ubiquity makes it difficult to determine the extent of their effects on genome evolution and gene expression because of the relative dearth of animal models lacking L1 activity. We have isolated L1 sequences from 11 megabat species by a method that enriches for recently inserted L1s and have done a bioinformatic examination of L1 sequences from a 12th species whose genome was recently shotgun sequenced. An L1 extinction event appears to have occurred at least 24 million years ago (MYA) in an ancestor of the megabats. The ancestor was unusual in having maintained two highly divergent long-term L1 lineages with different levels of activity, which appear, on an evolutionary scale, to have simultaneously lost that activity. These megabat species can serve as new animal models to ask what effect loss of L1 activity has on mammalian genome evolution and gene expression.

Extinction of LINE-1 activity coincident with a major mammalian radiation in rodents

LINE-1 transposable elements (L1s) are ubiquitous in mammals and are thought to have remained active since before the mammalian radiation. Only one L1 extinction event, in South American rodents in the genus Oryzomys, has been convincingly demonstrated. Here we examine the phylogenetic limits and evolutionary tempo of that extinction event by characterizing L1s in related rodents. Fourteen genera from five tribes within the Sigmodontinae subfamily were examined. Only the Sigmodontini, the most basal tribe in this group, demonstrate recent L1 activity. The Oryzomyini, Akodontini, Phyllotini, and Thomasomyini contain only L1s that appear to have inserted long ago; their L1s lack open reading frames, have mutations at conserved amino acid residues, and show numerous private mutations. They also lack restriction site-defined L1 subfamilies specific to any species, genus or tribe examined, and fail to form monophyletic species, genus or tribal L1 clusters. We determine here that this L1 extinction event occurred roughly 8.8 million years ago, near the divergence of Sigmodon from the remaining Sigmodontinae species. These species appear to be ideal model organisms for studying the impact of L1 inactivity on mammalian genomes.

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H2 uptake positive and H2 uptake negative strains were compared. None of the strains ofR. japonicum with high H2 uptake activities exhibited discernible plasmids, while most of the strains, with little or no H2 uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x106. An examination of H2 uptake negative mutants derived from an H2 uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.

A hydroxyproline-rich bacterial agglutinin from potato: its localization by immunofluorescence

Abstract Potato tubers (cv. Katahdin) contain a hydroxyproline-rich glycoprotein (HPRG) that agglutinates certain avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum . This and similar agglutinins are thought to play an important role in the immobilization of incompatible bacteria in potato and tobacco tissues. The agglutinin from potato tubers was purified by ion exchange chromatography. Antisera to the intact or deglycosylated agglutinin were obtained from New Zealand white rabbits after multiple intradermal and intramuscular injections. Immunoglobulins were precipitated with (NH 4 ) 2 SO 4 and antibodies specific for the agglutinin were purified by affinity chromatography. Frozen sections of petiole or leaf tissue from tobacco and potato were treated firstly with sheep normal immunoglobulin and then with either anti-agglutinin antibodies or normal rabbit immunoglobulin for 20 min. The sections were rinsed and then treated with fluorescein isothiocyanate-conjugated sheep anti-rabbit immunoglobulin. When the sections were examined by fluorescence microscopy, it was determined that anti-agglutinin antibodies bound only to the cell walls, particularly those of parenchyma. Fluorescence was also evident on the cell walls of tobacco and potato xylem vessels, epidermis, and collenchyma. Control sections treated with normal rabbit immunoglobulin did not bind the labelled anti-rabbit immunoglobulin. Cell walls in tissue sections from non-solanaceous plants such as soybean, corn, or begonia, treated in the same manner, were also stained by the labelled antibodies. Antibodies to both intact and deglycosylated potato agglutinin bound to these plant cell walls, indicating that the receptors are proteins with antigenic determinants which are similar to those of proteins from potato or tobacco cell walls. Such proteins (HPRGs) are common components of plant cell walls and may play a role in immobilizing bacteria that gain access to the intercellular spaces.

X Chromosome Inactivation and Xist Evolution in a Rodent Lacking LINE-1 Activity

Dosage compensation in eutherian mammals occurs by inactivation of one X chromosome in females. Silencing of that X chromosome is initiated by Xist, a large non-coding RNA, whose coating of the chromosome extends in cis from the X inactivation center. LINE-1 (L1) retrotransposons have been implicated as possible players for propagation of the Xist signal, but it has remained unclear whether they are essential components. We previously identified a group of South American rodents in which L1 retrotransposition ceased over 8 million years ago and have now determined that at least one species of these rodents, Oryzomys palustris, still retains X inactivation. We have also isolated and analyzed the majority of the Xist RNA from O. palustris and a sister species retaining L1 activity, Sigmodon hispidus, to determine if evolution in these sequences has left signatures that might suggest a critical role for L1 elements in Xist function. Comparison of rates of Xist evolution in the two species fails to support L1 involvement, although other explanations are possible. Similarly, comparison of known repeats and potential RNA secondary structures reveals no major differences with the exception of a new repeat in O. palustris that has potential to form new secondary structures.

Retrofitting the Genome: L1 Extinction Follows Endogenous Retroviral Expansion in a Group of Muroid Rodents

ABSTRACT Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes. Our previous demonstration that L1s and B1 short interspersed nuclear elements (SINEs) are inactive in a group of South American rodents led us to ask if other elements have amplified to fill the empty niche. We identified a novel and highly active family of ERVs (mysTR). To determine whether loss of L1 activity was correlated with expansion of mysTR, we examined mysTR activity in four South American rodent species that have lost L1 and B1 activity and four sister species with active L1s. The copy number of recent mysTR insertions was extremely high, with an average of 4,200 copies per genome. High copy numbers exist in both L1-active and L1-extinct species, so the mysTR expansion appears to have preceded the loss of both SINE and L1 activity rather than to have filled an empty niche created by their loss. It may be coincidental that two unusual genomic events—loss of L1 activity and massive expansion of an ERV family—occur in the same group of mammals. Alternatively, it is possible that this large ERV expansion set the stage for L1 extinction.

MysTR: an Endogenous Retrovirus Family in Mammals That Is Undergoing Recent Amplifications to Unprecedented Copy Numbers

ABSTRACT A large percentage of the repetitive elements in mammalian genomes are retroelements, which have been moved primarily by LINE-1 retrotransposons and endogenous retroviruses. Although LINE-1 elements have remained active throughout the mammalian radiation, specific groups of endogenous retroviruses generally remain active for comparatively shorter periods of time. Identification of an unusual extinction of LINE-1 activity in a group of South American rodents has opened a window for examination of the interplay in mammalian genomes between these ubiquitous retroelements. In the course of a search for any type of repetitive sequences whose copy numbers have substantially changed in Oryzomys palustris, a species that has lost LINE-1 activity, versus Sigmodon hispidus, a closely related species retaining LINE-1 activity, we have identified an endogenous retrovirus family differentially amplified in these two species. Analysis of three full-length, recently transposed copies, called mysTR elements, revealed gag, pro, and pol coding regions containing stop codons which may have accumulated either before or after retrotransposition. Isolation of related sequences in S. hispidus and the LINE-1 active outgroup species, Peromyscus maniculatus, by PCR of a pro-pol region has allowed determination of copy numbers in each species. Unusually high copy numbers of approximately 10,000 in O. palustris versus 1,000 in S. hispidus and 4,500 in the more distantly related P.maniculatus leave open the question of whether there is a connection between endogenous retrovirus activity and LINE-1 inactivity. Nevertheless, these independent expansions of mysTR represent recent amplifications of this endogenous retrovirus family to unprecedented levels.

Deletion mapping of H-Y antigen to the long arm of the human Y chromosome

A gene encoding or controlling the expression of the H-Y transplantation antigen was previously mapped to the human Y chromosome. We now report the sublocalization of this gene on the long arm of the human Y chromosome. Eight patients with Y-chromosomal abnormalities were examined with a series of existing and new DNA markers for the Y chromosome. The resulting deletion map was correlated with H-Y antigen expression. We conclude that the H-Y antigen gene maps to a portion of deletion interval 6 that is identified by specific DNA markers.

Molecular analysis of 46,XY females and regional assignment of a new Y-chromosome-specific probe.

SummaryThe relationship between Y-chromosome abnormalities and gonadal differentiation was investigated in six phenotypic females with a 46,XY karyotype and one patient with ambiguous genitalia secondary to apparently nonmosaic 46,XY mixed gonadal dysgenesis. No alterations were found in the Y chromosomes of six of these individuals by the use of either cytogenetic or molecular techniques. Cytogenetic analysis with high-resolution G-banding and Q-banding revealed a small deletion in the short arm of the Y chromosome in one female patient with some features of Turner syndrome. Southern hybridization with Y-specific probes showed a loss of DNA within deletion intervals 1, 2, and 3 of the Y chromosome. A new Y-chromosome-specific DNA probe that hybridizes to deletion interval 3 is described.

THE PRESENT STATUS OF HYDROGEN RECYCLING IN LEGUMES

ABSTRACT A brief discussion is presented of recent information concerning (a) factors influencing extent of N2, loss during N2 fixation by legumes; (b) electron carriers involved in the oxyhydrogen reaction of Rhizobium japonicum bacteroids; and (c) progress made in evaluating H2 recycling advantages. A major factor determining whether H2 is evolved from legume nodules is the presence of an active uptake hydrogenase which participates in the oxidation of H2 that is evolved as a by-product of the nitrogenase reaction. The extent of H2 evolution from the nitrogenase reaction is affected by those factors that influence the nitrogenase turnover rate. These include the supply of ATP and reductant and the ratio of the Fe protein to the MoFe protein component of nitrogenase. Oxidation of H2 in Rhizobium bacteroids is catalyzed by a series of enzymes located in bacteroid membranes. In addition to the hydrogenase per se, carriers so far shown to be involved in the process include cytochromes of the b and c types a...