Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.

2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

https://www.pnas.org/content/pnas/93/25/14862.full.pdf

Nucleotide sequence of the kaposi sarcoma-associated herpesvirus (hhv8)

Large-scale sequence analysis of the AD169 strain of human cytomegalovirus (HCMV) began in this laboratory in 1984 when very little was known about the sequence or location of genetic information in the viral genome. At that time sequence analysis was confined to the major immediate-early gene (Stenberg et al. 1984), a region of the Colburn strain that contained CA tracts (Jeang and Hayward 1983), the L-S junction region (Tamashiro et al. 1984), and what has been termed the transforming region (Kouzarides et al. 1983). This chapter is being written in March 1989 when the sequence is complete except for some remaining polishing of certain areas which is still going on (manuscript in preparation). As far as we know there are no major discrepancies in the data which might lead to the sequence changing although of course this cannot be ruled out. We present a preliminary analysis of the HCMV genome and limit ourselves mainly to the potential protein-coding content of over 200 reading frames.

Analysis of the Protein-Coding Content of the Sequence of Human Cytomegalovirus Strain AD169

A human cytomegalovirus (HCMV) glycoprotein gene with homology to glycoprotein B (gB) of herpes simplex virus and Epstein-Barr virus and gpII of varicella zoster virus has been identified by nucleotide sequencing. The gene has been expressed in recombinant vaccinia virus and the gene product recognized by monoclonal antibodies and human immune sera. Rabbits immunized with the recombinant vaccinia virus produced antibodies that immunoprecipitate gB from HCMV-infected cells and neutralize HCMV infectivity in vitro. These data demonstrate a role for this protein in future HCMV vaccines.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1986.tb04606.x

Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

Publisher Summary This chapter discusses random cloning and sequencing by the M13/dideoxynucleotide chain termination method. The dideoxy chain terminator/Ml3 vector method of deoxyribonucleic acid (DNA) sequencing is considered to be the fastest method to determine the sequence of large fragments of DNA. Lengths of DNA are cloned into the bacteriophage M13 that provides a source of large quantities of single-stranded DNA. This single-stranded DNA can then be used as a template in a primer extension dideoxynucleotide sequence reaction. The chapter discusses the problems related to the simplest method of breaking a DNA molecule into subfragments, which uses restriction endonucleases. The easiest method of purifying the fragment to be sequenced from the cloning vector is cleavage with suitable restriction endonuclease(s) followed by fractionation of the restricted DNA and isolation from a low gelling-temperature agarose minigel. The chapter discusses several procedures, such as isolation of fragment, fragment self-ligation, sonication, and others. Regarding size selection for DNA purification, two factors determine the minimum size to be selected. First, the insert to be sequenced should be at least as long as the maximum, which can be read from a normal sequence gel run. Second, as the subfragments have ends generated at random and not at specific primary sequence sites as with restriction enzymes, it is not possible to detect the junctions of religated noncontiguous fragments simply by inspection. M13 is the vector of choice for dideoxy sequencing for two main reasons. First, M13 bacteriophages are packaged in single strands of DNA, which are extruded from infected Escherichia coli cells into the surrounding culture medium. This means that considerable quantities of single-stranded template DNA can be easily produced. Second, the M13 mp vectors have a quick color assay to identify bacterial cells infected with phage containing an insert.

Random cloning and sequencing by the M13/dideoxynucleotide chain termination method.

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barrmore » virus.« less

Kathleen Weston

Papers

Analysis of the Protein-Coding Content of the Sequence of Human Cytomegalovirus Strain AD169

Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

Random cloning and sequencing by the M13/dideoxynucleotide chain termination method.

Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene.

Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus.