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Figure 3

Forms of human and mouse RBaK translated require an intact Linker motif to interact with GST-RB. autoradiograph showing that [S]methionine-labeled human RBaK transcribed and translated interacts with GST-RB () but not with GST alone (). autoradiograph of [S]methionine-labeled forms of mouse RBaK and human RBaK that were transcribed and translated , incubated with GST () or GST-RB ()-Sepharose beads, and analyzed by SDS-polyacrylamide gel electrophoresis. Input represents one-tenth of the volume of reticulocyte lysate incubated with GST protein. schematic diagram showing mutated forms of RBaK used in and .

Because there are no known RB-interacting motifs in RBaK proteins, we next sought to determine which motifs of mouse and human RBaK are required for RB binding. In order to determine which defined region of RBaK (the KRAB motif, zinc fingers, or linker region) was required for binding to RB, we performed a series of in vitro binding experiments using different forms of RBaK. Different lengths of the mouse and human RBaK were transcribed and translated in vitro to generate proteins containing the KRAB and linker (KL), the KRAB, and the first half of the linker (KΔL), or the linker region and several zinc fingers (LZ) (Fig. Exclusive Bonafini Collection Black oxford matte shoes Bonafini Cheapest bR8Cc
B, lanes 1–4 and 13 ). These translated proteins were then assayed for their relative ability to bind to GST or GST-RB in vitro . Like full-length human RBaK (Fig. Perfect Pay With Visa Cheap Online Converse Flyknit High Top Sneaker In China For Sale Best Authentic VAGKw3PH
A ), both mouse and human RBaK-KL bound well to GST-RB but not to GST alone (Fig. 3 B, lanes 5, 6, 9 , and 10 ); therefore, the zinc fingers were not required for binding to GST-RB in this assay. However, when the carboxyl-terminal half of the linker region was removed, the binding of both mouse and human RBaK-KΔL to GST-RB was greatly reduced (Fig. 3 B , compare lanes 8 and 12 with lanes 6 and 10 ), a finding indicating that an intact linker motif is required for both mouse and human RBaK-KL binding to GST-RB. Finally, in vitro translation of the linker and several zinc fingers from human RBaK (hRBaK-LZ) yielded a peptide that bound GST-RB with an affinity approximating that of RBaK forms retaining the KRAB domain (Fig. 3 B, lanes 13–15 ; some data not shown). Together, these results indicate that neither the KRAB motif nor the zinc fingers are required for GST-RB binding in this assay, whereas an intact linker motif may be required (Fig. 3 C ).

Having confirmed that both mouse and human forms of RBaK can interact with GST-RB in vitro and in the yeast two-hybrid system, we next began to characterize the functional properties of RBaK in vivo . After a plasmid containing the CMV promoter driving the expression of hRBaK was transfected into mouse 10T1/2 fibroblasts, mouse antiserum to human RBaK detected the expression of an ∼80-kDA protein. When cells were transfected with mutated RBaK cDNA (which generates Myc-tagged RBaK-KL), the anti-hRBaK antisera detected a 55-kDa protein instead of the ∼80-kDa protein (Fig. 4 A, lanes 1–3 ). Neither protein was detected in vehicle-transfected cells (data not shown). The ∼80-kDa protein detected in the presence of full-length RBaK cDNA was similar in size to the protein obtained by using in vitro transcription and translation (Fig. 3 A ), a finding consistent with the open reading frame predicted by the sequence analysis. The additional ∼85-kDa protein present in every lane may be an endogenous protein that is detected nonspecifically (Fig. 4 A ).

The second man who remembered seeing Klosowski in the basement shop was none other than Inspector Abberline, who was in charge of the hunt for Jack-the-Ripper. In the autumn of 1888, the Whitechapel area of East

* In order to apply to the university he needed to provide evidence of his birth, education, and training as a feldscher, together with character references. This collection of certificates were still in his possession when he was arrested in 1902, and it is from these that his early life can be reconstructed, even to knowing that while in Praga he lived at 16 Muranovskaja Street.

London saw a series of murders of prostitutes in which their bodies were badly mutilated. Just how many murders can be attributed to Jack-the-Ripper is debatable but in the autumn of that year six women met their deaths. The first murder actually occurred on a landing of George Yard Buildings, a stone's throw from the shop where Klosowski worked. The woman, 35-year-old Martha Turner, was stabbed 39 times with two knives in the early hours of the morning following Bank Holiday Monday, 6 August 1888. That Klosowski was the infamous murderer is doubtful but there were good grounds for suspecting him, and he invariably comes into the frame when the Ripper murders are discussed, mainly on the grounds that Inspector Abberline was convinced he was the man. This speculation does not really add to the story of him as an antimony poisoner, however.

Klosowski may eventually have taken over the hairdressing business but by June of 1889 he had his own barber's shop in Cable Street near the docks and had reverted to his real name of Severin Klosowski . He was becoming more sociable and while attending the Polish Club in St John's Square in Clerkenwell he met Lucy Baderski, a woman who came from the German-occupied part of Poland. Klosowski had little trouble getting Lucy to live with him; by the August Bank Holiday of that year she was telling her brother that they were married and she moved into the flat above the shop in Cable Street. In fact Klosowski and Lucy were not married until 23 October, when Lucy had become pregnant . As soon as Lucy and the baby were fit to travel, they set off for the USA in May 1890 and there Severin opened a barber's shop in New Jersey.

Perhaps this time the shop was a success, but that was certainly not true of his family life: rows between Lucy and Klosowski led to violence. By February of the following year Lucy had had enough and although she was then six months pregnant was prepared to brave the rough seas of a winter crossing of the Atlantic to return to London with their baby son. She went to live with her sister Mary at Scarborough Street, where on 12 May 1891 she gave birth to a baby girl.

Two weeks later her husband returned from the USA, and they were reconciled. Mary was persuaded to find alternative accommodation and moved out so that the family could be together, but the reconciliation did not last long and soon Lucy and Klosowski agreed to part permanently with the children staying with their mother. Although they never lived together again, they never got divorced.

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