MluI Fragment Research Articles

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44 66 (67%) of these clones crosshybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested ( n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of >370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3–4 Mbp.

Identification of proteins encoded in Escherichia coli hydA, hydB and analysis of the hydA locus

The hydB gene of Escherichia coli, which is related with the expression of hydrogenase activity, was cloned into the plasmid (pES1). Using the maxicell protein-labeling method, the molecular weight of hydB gene product was estimated. Comparing between the gene products from the mutant strains and that of the hydB genes cloned strains, the molecular weight of the gene product was 35 000 M r. Similarly, the molecular weight of the gene product of hydA, which had been previously cloned, was determined by maxicell analysis. The molecular weight of hydA gene product was estimated to be 80 000 M r. Using deletion analysis and Tn 1000 insertional inactivation of hydA's function, the hydA coding region was estimated between 2.2 kb and 2.8 kb in a 3.1 kb EcoRI- MluI fragment on the recombinant plasmid pEH3.

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.

Mega base map of the epidermal growth factor (EGF) receptor gene flanking regions and structure of the amplification units in EGF receptor-hyperproducing squamous carcinoma cells.

We have established a mega base scale physical map of the 5′‐ and 3′‐ flanking regions of the epidermal growth factor (EGF) receptor gene using CpG‐recognition rare‐cutting restriction enzymes and pulsed‐field gel electrophoresis. In this map, a methylation‐free CpG island (HTF island) is located within an 8‐kilobase pair (kb) EcoRI fragment which includes exon 1 of the EGF receptor gene. From this HTF island, a 390‐kb NotI fragment was identified as the longest 5’‐flanking region and a 540‐kb MluI fragment as the longest 3′‐flanking region. Utilizing this map information, we have analyzed the structure of the flanking regions of amplified EGF receptor genes which are found in various squamous carcinoma cells. Among seven cell lines tested, four cell lines carrying EGF receptor genes in amounts more than 20 times that of normal cells showed amplification together with large 5′‐ and 3′‐ flanking regions. The amplified 5′‐flanking regions were rearranged in different forms but were distinct in each cell line. The amplified 3′‐flanking regions were at least 540 kb in size and common to all the cell lines, except that A431 had rearrangement points within 540 kb downstream of the HTF island. Thus, the size of amplification units appears to be large and different in each cell line.

RNA-mediated gene conversion?

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.

Cloning of the glucokinase gene in Escherichia coli B.

A gene for glucokinase (Glk) in Escherichia coli B was cloned onto vector plasmid pBR322, and the hybrid plasmid obtained was designated pGK100. The gene for Glk was located in the central MluI fragment (0.82 megadalton) of the 6.0-megadalton chromosomal DNA inserted in the HindIII site of the vector. The introduction of pGK100 into E. coli 112L having a decreased level of Glk activity resulted in the about 15-fold increase in this enzyme activity. The poor growth rate of 112L cells on glucose or mannose was also improved by the introduction of pGK100. However, removal of some portion near the glk gene prevented the growth of 112L cells, although Glk activity was high enough to support growth. Therefore, some function of Glk may be regulated by a gene(s) near the glk gene.